STUDY OF TRAUMATIC BRAIN INJURY BIOMARKERS BY MASS SPECTROMETRY

By

MANASI N. KAMAT

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2015

© 2015 Manasi N. Kamat

To my family

4

ACKNOWLEDGMENTS

First, I would like to thank my advisor, Dr. Richard A. Yost, for letting me join his

group. The discussions and suggestions were valuable to improve my work. Then, I

would like to thank my collaborator and mentor, Dr. Kevin K.W. Wang, for giving me the

opportunity to work with his group. His guidance helped me build up my interest in the

field of proteomics.

Next, I would like to thank my co-advisor, Dr. David H. Powell, for allowing me

perform my research and also allowing me to work as a teaching assistant in his mass

spectrometry service facility. I would also like to thank Dr. Kari Basso, who later joined

the mass spectrometry service facility, for letting me continue my research and teaching

assistantship in the same mass spectrometry service facility. She also helped me with

my research by giving some valuable suggestions. I would like to acknowledge my

committee member Dr. Timothy Garrett for the discussion we had during my oral

qualifying exam.

I would like to thank all the past and current members of the Yost group. The

discussions with them have helped me improve in all aspects during my graduate

career. I would like to acknowledge Dr. Ahmed Moghieb for the valuable suggestions

and also for helping train me on instrument handling. I would like to thank Dr. Zhihui

Yang for arranging all the mouse brain samples. I would also like to acknowledge Dr.

Firas Kobeissy, George Sarkis, Carl Bauer and Dhwani Kumar for all their help.

Finally, I would like to thank all my friends and family. They have been very

supportive throughout my graduate study, most of all, my husband Naren, for all the

support and encouragement to pursue this career.

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TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES ............................................................................................................ 7

LIST OF FIGURES .......................................................................................................... 8

ABSTRACT ................................................................................................................... 12

CHAPTER

1 BACKGROUND ...................................................................................................... 14

Introduction ............................................................................................................. 14 Mass spectrometry based proteomics .................................................................... 15

Ionization Techniques ...................................................................................... 16 Mass Analyzers ................................................................................................ 17 Separation Techniques .................................................................................... 18

Western Blot ........................................................................................................... 19 Sample Preparation .......................................................................................... 19

Gel Electrophoresis .......................................................................................... 20 Blotting ............................................................................................................. 21 Blocking and Antibody incubation ..................................................................... 22

Detection .......................................................................................................... 22

2 IDENTIFICATION OF BIOMARKERS FOLLOWING TRAUMATIC BRAIN INJURY BY PERFORMING IN VITRO DIGESTION STUDY .................................. 30

Introduction ............................................................................................................. 30

Experimental ........................................................................................................... 31 Biological Sample Preparation ......................................................................... 31 Digestion of Proteins ........................................................................................ 33

Instrumentation ................................................................................................. 34 Results and Discussion........................................................................................... 36

Mass spectrometry ........................................................................................... 36 Western Blot ..................................................................................................... 39

Conclusions ............................................................................................................ 41

3 IDENTIFYING PROTIEN BIOMARKERS IN CLINICAL HUMAN CEREBROSPINAL FLUID (CSF) SAMPLES .......................................................... 71

Introduction ............................................................................................................. 71 Experimental ........................................................................................................... 71

Cerebrospinal fluid (CSF) samples ................................................................... 71

6

Sample preparation .......................................................................................... 72

Instrumentation ....................................................................................................... 73 Results and Discussion........................................................................................... 74

Mass spectrometry ........................................................................................... 74 Western blot ..................................................................................................... 76

Conclusions ............................................................................................................ 77

4 SYSTEMS BIOLOGY APPROACH TRAUMATIC BRAIN INJURY BIOMARKERS ........................................................................................................ 90

Introduction ............................................................................................................. 90 Pathway analysis .................................................................................................... 90 Experimental ........................................................................................................... 91 Results and Discussion........................................................................................... 91

Conclusions ............................................................................................................ 93

5 SUMMARY AND FUTURE DIRECTIONS .............................................................. 97

Summary ................................................................................................................ 97 Future Work ............................................................................................................ 98

LIST OF REFERENCES ............................................................................................. 102

BIOGRAPHICAL SKETCH .......................................................................................... 105

7

LIST OF TABLES

Table page 2-1 List of protein biomarkers identified by performing in vitro digestion of mouse

brain lysate using LC-MS/MS. ............................................................................ 46

2-2 List of protein biomarkers identified in CCI mouse brain lysate by LC-MS/MS. .. 58

3-1 List of protein biomarkers identified in human CSF samples by LC-MS/MS. ...... 80

4-1 List of brain injury protein biomarkers entities .................................................... 94

5-1 Summary of biomarkers identified by invitro digestion, mouse CCI samples, and human TBI CSF samples. .......................................................................... 100

8

LIST OF FIGURES

Figure page 1-1 Nomenclature for peptide fragments generated by tandem mass

spectrometry. (Adapted from Paizs and Suhai) [9] ............................................. 24

1-2 Schematic depiction of an ESI source. (Adapted from Konermann) [17] .............. 25

1-3 Representation of the MALDI process. (Adapted from Chughtai et al.) [37] ......... 26

1-4 Molecular weight markers used in gel electrophoresis. (Adapted from Life technologies) [38] ................................................................................................. 27

1-5 Schematic of gel electrophoresis. ....................................................................... 28

1-6 Schematic of transfer of separated protein form the gel to a solid support membrane (PVDF membrane). .......................................................................... 28

1-7 Schematic showing binding of primary and secondary antibodies to the bound proteins on the membrane. ...................................................................... 29

2-1 Calpain and excitotoxicity. Excessive glutamate levels during cerebral ischemia causing overactivation of ionotropic receptors allowing Ca2+/Na+ influx. .................................................................................................................. 43

2-2 Workflow of sample processing of mouse brain lysates (control and CCI). ........ 44

2-3 Venn diagram showing the number of proteins identified using Proteome Discoverer based on the LC-MS/MS data from digested naïve brain lysate of cortex (A), corpus callosum (B) and hippocampus (C) region. ........................... 45

2-4 Mouse neurogranin sequence with three mapped peptides, identified by database search result. ...................................................................................... 47

2-5 MS/MS spectra for the neurogranin peptide AAKIQASF. (A).MS/MS spectra displaying the fragment ions for this peptide. ..................................................... 47

2-6 Identified b- and y-type ions for the neurogranin peptide AAAAKIQASF shown in red and blue. ....................................................................................... 48

2-7 Identified b- and y-type ions for the neurogranin peptide GPGPGGPGGAGGARGGAGGGPSGD shown in red and blue. ....................... 49

2-8 Mouse BASP-1 sequence with two mapped peptides identified by database search result. ...................................................................................................... 50

2-9 MS/MS spectra for the BASP-1 peptide EAPAAAASSEQSV. (A) MS/MS spectra displaying the fragment ions for this peptide. ......................................... 51

9

2-10 Identified b- and y-type ions for the BASP1 peptide EAPAAAASSEQSVAVKE shown in red and blue. ....................................................................................... 52

2-11 Identified b- and y-type ions for the BASP1 peptide AEPAPSSKETPAASEAPSS shown in red and blue. ......................................... 53

2-12 Mouse MBP isoform sequence with two mapped peptides identified by database search result. One is marked in red and the second one is underlined. .......................................................................................................... 54

2-13 MS/MS spectra for the MBP peptide KNIVTPRTPPP. (A) MS/MS spectra displaying the fragment ions for this peptide. ..................................................... 55

2-14 Identified b- and y-type ions for the MBP peptide LATASTMDHAR shown in red and blue........................................................................................................ 56

2-15 Venn diagram showing the number of proteins identified using Proteome Discoverer based on the LC-MS/MS data from CCI brain lysate samples of cortex (A), corpus callosum (B) and hippocampus (C) region. ........................... 57

2-16 Western blot of the digested naïve brain lysate of cortex region probed with alpha-spectrin antibody. ..................................................................................... 59

2-17 Western blot of the digested naïve brain lysate of corpus callosum (CC) region probed with alpha-spectrin antibody.. ...................................................... 60

2-18 Western blot of the digested naïve brain lysate of hippocampus region probed with alpha-spectrin antibody. .................................................................. 61

2-19 Western blot of the digested naïve brain lysate of Cortex region probed with GFAP antibody displaying the intact and breakdown product (38 kDa). ............. 62

2-20 Western blot of the digested naïve brain lysate probed with MBP antibody (A) Western blot displaying the breakdown product of MBP. ................................... 63

2-21 Western blot of the digested naïve brain lysate of cortex region probed with neurogranin antibody. (A) Western blot displaying the breakdown product of neurogranin observed in the calcium and calpain digested samples. ................. 64

2-22 Western blot of the digested naïve brain lysate of corpus callosum region probed with neurogranin antibody. ..................................................................... 65

2-23 Western blot of the digested naïve brain lysate of hippocampus region probed with neurogranin antibody. ..................................................................... 66

2-24 Western blot of CCI brain lysates of all the three regions probed with alpha-spectrin antibody. (A) Western blot displaying the breakdown product of spectrin observed CCI brain lysate samples....................................................... 67

10

2-25 Western blot of CCI brain lysates of all the three regions probed with GFAP antibody. ............................................................................................................. 68

2-26 Western blot of CCI brain lysates of all the three regions probed with MBP antibody. (A) Western blot displaying the breakdown product of MBP observed CCI brain lysate samples. ................................................................... 69

2-27 Western blot of CCI brain lysates of all the three regions probed with neurogranin antibody. (A) Western blot displaying the intact neurogranin observed CCI brain lysate samples. ................................................................... 70

3-1 Venn diagram showing the number of proteins identified using Proteome Discoverer based on the LC-MS/MS data from human clinical TBI CSF samples. ............................................................................................................. 79

3-2 Human MBP isoform sequence with mapped peptides identified from database search results. .................................................................................... 81

3-3 MS/MS spectra for the MBP peptide HGSKYLATASTMD. (A) MS/MS spectra displaying the fragment ions for this peptide. ..................................................... 82

3-4 Identified b- and y-type ions for the MBP peptide TQDENPVVHF shown in red and blue........................................................................................................ 83

3-5 Human neurogranin sequence with the mapped peptide identified from database search results. .................................................................................... 84

3-6 MS/MS spectra for the neurogranin peptide GPGGPGGAGVARGGAGGGP. (A) MS/MS spectra displaying the fragment ions for this peptide. ...................... 85

3-7 Western blot of human TBI-CSF samples, that were retained on the 10,000Da molecular weight cut off (MWCO) filter after filtration and probed with alpha-spectrin antibody. .............................................................................. 86

3-8 Western blot of human TBI-CSF samples, that were retained on the 10,000Da molecular weight cut off (MWCO) filter after filtration and probed with GFAP antibody. ........................................................................................... 87

3-9 Western blot of human TBI-CSF samples, that were retained on the 10,000Da molecular weight cut off (MWCO) filter after filtration and probed with MBP antibody. ............................................................................................. 88

3-10 Western blot of human TBI-CSF samples, that were retained on the 10,000Da molecular weight cut off (MWCO) filter after filtration and probed with neurogranin antibody. ................................................................................. 89

4-1 Mapping of protein biomarkers showing direct interaction pathway. ................... 95

11

4-2 Mapping of protein biomarkers showing regulations of cell process and diseases. ............................................................................................................ 96

5-1 Protein degradome and its localization. ............................................................ 101

12

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

STUDY OF TRAUMATIC BRAIN INJURY BIOMARKERS BY MASS SPECTROMETRY

By

Manasi N. Kamat

December 2015

Chair: Richard A. Yost Major: Chemistry

Traumatic brain injury (TBI) is a serious health problem affecting more than 1.7

million people each year in the United States. TBI results from a jolt or penetration of an

object into the head. TBI can have a range of physical and psychological effects. Some

symptoms of TBI can be observed immediately following the traumatic event, while

some may be seen after a few days or few weeks. TBI symptoms depend on the

severity of the injury. The symptoms include headache, unconsciousness, blurred

vision, mood swings, concussions; loss of memory and in extreme cases, death.

Neurotrauma following TBI promotes degradation of proteins in the brain. One of

the degradation pathways involves excitatory amino acids (glutamate and aspartate)

and their receptors. Excessive synaptic level of glutamate is neurotoxic, activating

specific receptors causing an influx of Na+ and Ca2+ cations into the cytosol. Excess

influx of Ca2+ into the cytosol activates calpain, a calcium-dependent system, which

cleaves cytoskeletal proteins. These breakdown products can serve as biomarkers for

TBI that can be used to study post-injury mechanism. This research investigated the

breakdown products of proteins in pathological samples from TBI using mass

spectrometry.

13

The research presented here focused on identifying potential protein biomarkers

in an animal TBI model, and then correlated the data to clinical human CSF samples

using LC-MS/MS. The first project focused on studying in vitro digestion of the naïve

brain lysate with calpain-1, used as a protease to study the calpain-activated breakdown

of proteins. The list of potential protein biomarkers obtained from this in vitro digestion

was used to identify protein biomarkers from the controlled cortical impact (CCI) injury

(an animal TBI model) brain lysate samples. In the second project, the same

methodology was used for human cerebral spinal fluid (CSF) samples to identify

potential calpain activated breakdown products of proteins. The third project presents a

system biology approach to determine the biochemical pathway involved in the

pathogenesis of brain injury based on the protein biomarkers identified by in vitro

digestion of brain lysate, animal TBI model, and human clinical CSF samples.

14

CHAPTER 1 BACKGROUND

Introduction

Proteins are large, complex molecules that play a critical role in the body. [1]

They are made up of one or more long chains of amino acids. [2] The sequence of

amino acids determines each protein structure and its function. [3] Proteins perform a

large range of functions in the body. They are called the building blocks of the body.

They provide structure to the body, like collagen, which is found in various connective

tissues that provide framework for the ligaments to hold bones together and the tendons

that attach muscles to those bones. Proteins also regulate body processes. They act as

enzymes, which catalyze chemical reactions. Some enzymes act on other proteins by

adding or removing functional groups that is called post-translational modification

(PTM). [3] Hormones, which are proteins, help to coordinate certain bodily activities. A

very commonly known example, insulin, is a hormone, regulates sugar levels in the

blood. Proteins also function as transport materials. Hemoglobin, which is an oxygen

transporting protein found in blood cells is such a type of transport material. Proteins

also function as antibodies that are involved in defending the body from foreign

invaders.

Proteomics is the study of proteins that involves identification, localization, and

functional analysis of the protein make-up of the cell. The term proteome refers to the

complement of proteins, including modifications made to a particular set of proteins,

produced by an organism or a cellular system. [4] It is known that mRNA is not always

translated into protein, and the amount of protein produced for a given amount of mRNA

depends on the gene it is transcribed from and on the current physiological state of the

15

cell. [3] Recently, scientists are more interested in proteomics because it gives much

better understanding of an organism than genomics. The gene expression involves

transcription of genes, which is then followed by translation of messenger RNA (mRNA)

to produce proteins. The proteome is much more complex than the genome because

each protein can be chemically modified in different ways after synthesis. [1] The field of

proteomics is particularly important because most diseases are manifested at the level

of protein activity.

Mass spectrometry based proteomics

Mass spectrometry (MS)-based proteomics has emerged as a powerful

technique due to advances in instrumentation, sample preparation and computational

tools. [5, 6] Most of the proteomics work is done using tandem mass spectrometry

(MS/MS). [7] The common approach is bottom-up proteomics. [7] In bottom-up

proteomics, the proteins extracted from cell or tissue lysate are digested by protease to

smaller peptides. These peptides are then separated by liquid chromatography and then

analyzed by MS/MS to sequence the peptides. [8] The MS/MS data acquisition consists

of two stages. The first stage involves analyzing all the peptide ions that are introduced

into the instrument at a given time (MS spectrum). In the second stage, selected peptide

ions, (precursor ions) are fragmented into smaller pieces in the collision cell of the mass

spectrometer by a process called collision induced dissociation (CID). The

fragmentation of a peptide usually takes place at one of the bonds along the peptide

backbone. [9] On fragmentation six types of fragment ions can be generated that are

termed as a, b, c, x, y, and z type ions. The most common type of ions formed from the

CID energy is the b and the y type ions. [9, 10] As shown in Figure 1-1, the b type ion

extends from the amino terminus, also called as the N-terminus, and the y type ion

16

extends from the carboxyl terminus known as the C-terminus. The number in the

subscript denotes the number of amino acid remaining in the peptide fragment. [9] The

fragmentation pattern encoded by the MS/MS spectrum allows identification of the

amino acid sequence of the corresponding peptide. The MS/MS spectra are then

searched against protein sequence database for peptide identification. [11] A number of

automated database search tools are available. The peptides database search engines

such as MASCOT [12] , Sequest [13] , or X Tandem [14] can be used to identify the amino

acid sequence from the fragmented peptides. All these search engines operate in a

similar manner. The experimental MS/MS spectra are compared against theoretical

fragmentation patterns constructed for peptides from the searched database to find a

match. The best scoring peptide match has the highest likelihood of being correct.

Ionization Techniques

Mass spectrometry based proteomics has grown rapidly due to advances in

experimental methods, instrumentation, and data analysis. Electrospray ionization (ESI)

[15] and matrix-assisted laser desorption/ionization (MALDI) [16] are the two most

commonly used ionization techniques to ionize peptides and proteins for mass

spectrometric analysis. ESI is driven by high voltage (2–6 kV) applied between the

emitter at the end of the separation pipeline and the inlet of the mass spectrometer. ESI

involves creation of an electrically charged spray, which is known as the Taylor cone. It

is followed by the formation and desolvation of analyte-solvent droplets (Fig 1-2) [17]

Formation and desolvation of the droplets is aided by a heated capillary, and in some

cases, by a sheath gas flow at the mass spectrometer inlet. Multiply charged ion

species are formed by ESI. [18] One important development in ESI technique includes

micro and nano-ESI in which the flow rates are lowered to nanoliter per min to improve

17

the method’s sensitivity. As the flow rate is lowered, the dimensions of the Taylor cone

and the droplets that are produced are also reduced. Hence the efficiency of desorption

of analyte ions from the electrosprayed droplet increases as the size of the droplets

decreases because of the larger surface area of the droplet relative to its total volume.

As a result greater proportion of analyte is desorbed from the droplets and transmitted

to the mass analyzer. This increases the efficiency on the order of 500-fold. Due to

increase in efficiency, the detectable signals can be observed with attomole to

femtomole amounts of analyte. [6] A practical advantage of nanospray in proteomics is

that the microliter volumes of sample produced from protein digestion can be sprayed

for extended periods. Since the ions are generated for longer period, tandem mass

spectrometry can be performed to investigate the structure of product ions.

On the other hand, MALDI sublimates and ionizes the samples out of a dry,

crystalline matrix via laser pulses. [19] A MALDI matrix absorbs the laser energy and

transfers it to the analyte, where the laser heating causes desorption of matrix and

analyte ions into the gas phase (Fig 1-3). [20] MALDI generated ions are predominantly

singly charged ions, which makes MALDI applicable to top-down analysis of high

molecular weight proteins.

Mass Analyzers

Mass analyzers are integral part of each instrument since they can store and

separate the ions based on the mass to charge ratios. Ion trap, time-of-flight (TOF),

orbitrap and quadrupole are the most commonly used mass analyzers in proteomics

research. [21, 22] For structural analysis like peptide sequencing, two steps of mass

spectrometry are performed in tandem (MS/MS), which can be achieved by employing

the same separation principle twice (e.g. TOF/TOF) [23] or by combining two different MS

18

(Q-TOF) [24] separation principles. Since MALDI produces a short burst of ions in the

vacuum, it is mainly coupled with TOF MS that measures intact proteins or peptides.

Recently, MALDI ion sources have been coupled with quadrupole ion-trap MS [25] and

hybrid quadrupole TOF MS. [26] Electrospray produces a continuous beam of ions in

atmosphere and they are coupled with TOF, quadrupole and ion trap MS that are used

to generate product ion spectra of the precursor ions. [21, 22] In this research, all the work

was performed on Thermo Scientific LTQ XL, a linear ion trap coupled with Waters

NanoAcquity UPLC system.

Separation Techniques

Separation techniques simplify complex biological samples prior to mass

analysis. Because proteins are identified by the mass-to-charge ratios of their peptides

and fragments, sufficient separation is required for unambiguous identifications. [27] The

high-pressure liquid chromatography (HPLC) is usually directly coupled to instruments

with an electrospray ionization (ESI) source. Continuous separation by HPLC is

compatible with a continuous ionization source such as ESI. Hence both are interfaced

with scanning or trapping mass analyzers like a, LTQ. HPLC has been used commonly

in many biological samples. Reversed phase liquid chromatography (RPLC) separates

compounds based on their hydrophobicity. It has been shown that packing long, narrow

capillary RP columns greatly improves loading capacity, sensitivity, and dynamic range

of the RPLC. The small particle size of RP material (2 μm and smaller) allows improved

peak capacity, resolution, and reduced analysis time when using an ultrahigh pressure

system. The small particle size, the elevated temperature (65°C) and the ultra-high

performance liquid chromatography (UPLC) approach were shown to improve

separation of intact proteins. [28] Therefore, increasing the column length while

19

decreasing the particle size and using UPLC leads to improved peak capacity,

resolution, sensitivity, and analysis time. [29]

Western Blot

Western blot is a widely used technique for the detection of proteins based on

the ability to bind to specific antibodies. [30] Specific proteins can be identified from a

complex mixture of proteins. This analytical technique includes 3 stages: 1) separation

of proteins by its weight, 2) transfer of proteins to a solid support and 3) marking target

proteins with proper primary and secondary antibody for visualization. [30, 31]

Sample Preparation

First, the sample preparation involved isolation of proteins from a sample.

Proteins are extracted from tissues, cell, CSF, serum, etc. by lysis. The concentration of

protein is determined. Since the lysates is a mixture of proteins, they are then separated

by polyacrylamide gel electrophoresis (PAGE). [30, 31] Before the samples are loaded

onto the gel, they are denatured and reduced. The samples are mixed with loading

buffer called Laemmli buffer. The loading buffer contains sodium dodecyl sulfate (SDS),

2-mercaptoethanol, glycerol, bromophenol blue and tris-HCl. SDS denatures the protein

by wrapping around the polypeptide backbone. Thus, anionic SDS confers a negative

charge on the protein. [32] 2-mercaptoethanol reduces the disulfide bonds making the

protein to adopt random coil configuration. Glycerol in the loading buffer gives density to

the samples. This helps the sample to sink down to the bottom of the well. Bromophenol

blue is a small anionic dye that enables in visualization of the migration of proteins.

Since this compound is small, it will migrate through the gel faster than any of the other

components of the sample. This helps to provide a migration front to monitor the

progress of separation.

20

Gel Electrophoresis

Electrophoresis is a simple, and sensitive analytical tool used to separate

proteins based on their masses. Electrophoresis can be one-dimensional or two-

dimensional. The proteins are separated based on their molecular weight as they move

through the gel matrix in electrophoresis. [33] The gel matrices are of two types

polyacrylamide and agarose. When proteins are separated on polyacrylamide gels the

procedure is termed as SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel

electrophoresis). These gel matrices being porous act as a molecular sieve.

Polyacrylamide is formed by polymerization of acrylamide, a monomer molecule cross-

linked by N, N- methylenebisacrylamide, abbreviated as BIS. Ammonium persulfate

which generates free radicals and N, N, N’, N’- tetramethylethylene diamine (TEMED), a

catalyst, are added during polymerization since acrylamide and BIS are non-reactive

when mixed together. The pore size of the gel depends on the percentage of acrylamide

and cross-linker. As the amount of acrylamide increases the pore size decreases. Thus,

a higher percentage of acrylamide-BIS is used for separation of smaller proteins of

interest. [31]

The samples mixed with the sample buffer are loaded on to the gel. Molecular

weight markers are used to define the size of the proteins run in the gel. A marker is

added to at least one lane of the gel. A marker is composed of different proteins of

known size and also since they are visible progress of the proteins in the electrophoretic

run can be monitored. The distances migrated by the marker proteins over the time

course of the run provide a logarithmic scale by which the size of the unknown proteins

can be calculated. Figure 1-4 shows an image of separated visible full range and low

range molecular weight markers. The gel is then connected to a power supply and a

21

current is run across the gel. The gel is allowed to run for few hours in a buffer tank for

separation of proteins. Gel electrophoresis involves an electrical field such that the wells

of the gel have a negative charge and the opposite end is positively charged. The

proteins are not initially negatively charged, as mentioned previously. When treated with

SDS, the proteins unfold into a linear shape and are coated with negative charge. Thus

the negatively charged proteins migrate towards the positively charged end of the gel,

separating on their way. A schematic of gel electrophoresis is shown in Figure 1-5.

Blotting

After separation of proteins by gel electrophoresis, the proteins are transferred

from the gel to a solid support membrane. This membrane is of two types, nitrocellulose

and polyvinylidene fluoride (PVDF). Both of these membranes bound to proteins with

high affinity. Nitrocellulose membrane is brittle and this makes it less efficient for

reprobing. PVDF membrane has better mechanical support and can also be reprobed.

[34] Applying an electrical field perpendicular to the surface of the gel causes the transfer

of the proteins from the gel on to the membrane. It is very important to maintain a close

contact between the gel and the membrane for effective transfer of proteins. The gel

and the membrane are assembled in a sandwiched configuration with sheets of filter

papers to ensure close contact. The membrane is placed between the gel and positive

electrode so that when a current is applied, the negatively charged proteins migrate on

to the membrane. Transfer buffer is used for blotting which has same function as that of

running buffer in gel electrophoresis. Wet transfer and semi-dry transfer are two

methods of blotting. In a wet transfer, the sandwich of gel and membrane is placed in a

cassette, and then immersed in a tank filled with transfer buffer, and subjected to an

electrical field. It takes few hours for transfer to take place. Whereas in a semi-dry

22

transfer, the gel/membrane sandwich is placed on the electrode plates with minimal

transfer buffer, confined to the filter papers. This transfer takes about an hour. A

schematic showing transfer of proteins is shown in Figure 1-6.

Blocking and Antibody incubation

Blocking is important, as it prevents non-specific binding of the antibody to the

blotting membrane. Commonly, non-fat dried milk powder (5%) dissolved in a solution of

tris buffered saline tween (TBST) is used as blocking solution. Non-fat dried milk is used

widely used because it is inexpensive and available easily. Bovine serum albumin

(BSA) (5%) in TBST is also used as blocking solution. [35]

Following blocking, the membrane is incubated in a dilute solution of primary

antibody prepared in 5% milk with TBST for few hours at room temperature or overnight

at 4°C. The primary antibody used is specific to the target protein. The membrane is

then washed with TBST following incubation with the primary antibody to minimize the

background and also to remove any unbound antibody. The membrane is then

incubated in secondary antibody prepared in 5% milk in TBST for an hour at room

temperature. Selection of secondary antibody depends on the species in which the

primary antibody was produced. [32] For example, if the primary antibody is an IgG type

produced in goat, then the secondary antibody will be an anti-goat IgG antibody

produced in another species so that it will bind to the Fc region of the primary antibody.

The membrane is washed again with TBST.

Detection

Membrane bound proteins are generally detected using secondary antibodies

that are labeled with a radioisotope or a fluorophore or an enzyme alkaline phosphatase

(AP) or horseradish peroxidase (HRP). [32] Most common detection methods use

23

secondary antibodies conjugated to AP. [36] In this method when the enzyme substrate is

added, like 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium

(NBT), a colored precipitate is deposited. A schematic of antibody binding and detection

is shown in figure 1-7.

24

Figure 1-1. Nomenclature for peptide fragments generated by tandem mass spectrometry. (Adapted from Paizs and Suhai) [9]

25

Figure 1-2. Schematic depiction of an ESI source. (Adapted from Konermann) [17]

26

Figure 1-3. Representation of the MALDI process. (Adapted from Chughtai et al.) [37]

+

+

+ +

+

+ +

+

+

+

+

+

+

Stainless Steel Plate

Analyte/Matrix Mixture Laser Beam

Matrix Ion Analyte Ion

+

To Mass Analyzer

27

Figure 1-4. Molecular weight markers used in gel electrophoresis. (Adapted from Life

technologies) [38]

Low range marker

Full range rainbow marker

28

Figure 1-5. Schematic of gel electrophoresis.

Figure 1-6. Schematic of transfer of separated protein form the gel to a solid support

membrane (PVDF membrane).

-

+

Electrophoresis cell Gel Resolved Gel

Cathode (-)

Anode (+)

Blotting paper PVDF membrane

Gel

Blotting paper

29

Figure 1-7. Schematic showing binding of primary and secondary antibodies to the

bound proteins on the membrane.

PVDF Membrane

Target protein

Primary Antibody Secondary Antibody HRP conjugate

BCIP-NBT Substrate

Enzyme - Alkaline phosphatase

30

CHAPTER 2 IDENTIFICATION OF BIOMARKERS FOLLOWING TRAUMATIC BRAIN INJURY BY

PERFORMING IN VITRO DIGESTION STUDY

Introduction

Traumatic brain injury (TBI) is a serious health disorder that contributes to a

substantial number of deaths and disabilities each year. [39, 40] As per the data by the

Center for Disease Control (CDC), 1.7 million TBI cases are being reported every year.

Approximately 275,000 patients are hospitalized and about 52,000 die every year in the

US. [41] Currently, clinical data collected from the pathophysiological studies have

resulted in therapeutic targets, but improvement is still needed. There is a need for a

better understanding of the pathophysiology of the head injury for adequate treatment.

[42]

The brain injuries are classified into two types; focal injuries and diffuse injuries.

[43, 44] Focal injuries are caused by a direct blow to the head that results in laceration,

hemorrhage etc. Diffuse injuries are caused by an acceleration/deceleration type injury

that results in diffuse axonal injury; one of the mechanisms of TBI. [45] Neurological injury

does not occur immediately at the moment of impact, but evolves later on. The outcome

from the injury is determined by two stages; 1) primary insult, damage occurring at the

moment of impact and 2) secondary insult, which involves pathological processes

initiated with a delay following the moment of injury (minutes to months). A secondary

insult is an indirect result of the injury. [39, 46]

One of the pathophysiology processes involves excessive release of excitatory

neurotransmitters (glutamate and aspartate) causing membrane depolarization. [43] The

release of these excitatory neurotransmitters activates their receptors; N-methyl-D-

aspartate (NMDA) receptor and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

31

receptor (AMPA) coupled to a Na+ and Ca2+ ionophore, causing an influx of these

cations to the cytosol (figure 2-1). [43, 44, 47] An excessive synaptic level of these

neurotransmitters is neurotoxic. Na+ influx causes depolarization that opens voltage-

gated neuronal Ca2+ channels, resulting in further Ca2+ influx. Influx of these Ca2+ ions

causes activation of Ca2+-dependent system like calpain that further cleaves

cytoskeletal proteins. [43, 47] In this work, in vitro digestion was performed using naïve

brain lysate to study the breakdown of proteins following calpain proteolytic cleavage.

Based on the MS/MS data and database search engine, potential protein biomarkers

were identified. These proteins were then compared to the TBI brain lysates.

Experimental

Biological Sample Preparation

CB57BL/6 male mice, 3 to 4 months old (Charles River Laboratories, Raleigh,

NC) were used for this study. TBI in mice was induced using the controlled cortical

impact (CCI) technique. The mice were anesthetized using 4% isofurane in oxygen as a

carrier gas for 4 mins and maintained under anesthesia with 2-3% isofurane in oxygen.

Surgery was performed on the mice by mounting in a stereotactic frame in a prone

position and secured by ear and incisor bars. A 3 mm unilateral (ipsilateral) craniotomy

was performed midway between bregma and lambda with the dura mater remaining

intact over the cortex. A brain trauma was induced by using an Impact one (Leica

Biosystems) impactor by impacting the right cortex (ipsilateral cortex) with a 2mm

diameter impactor tip at a velocity of 3.5 m/second, 1.5mm compression depth, and a

200 ms dwell time (compression duration). Occasional bleeding resulting from the CCI

(typically due to a ruptured blood vessel), was controlled by either a sterile cotton tip

applicator or sterile gelfoam, which was removed before closing of the skin incision with

32

sterile wound clips, 4-0 Nylon (Ethilon) sutures or 4-0 Monocryl sutures. After closing of

the skin incision, animals were placed on disposable underpads in recovery cages that

are maintained at ~38°C by placing a regular electric heating pad kept at low

temperature underneath the recovery cages. Immediately after surgery, all mice were

injected with saline solution (volume = 3% of body weight) to reduce the risk of

anesthesia-induced hypothermia and dehydration. Animals typically recover from

anesthesia and CCI surgery in from 5 to 30 minutes. After recovery mice were placed

back in their home cage and kept under observation in the surgery room for at least two

hours before they are returned to the animal facility. After 24 hours the animals were

euthanized with a lethal dose of pentobarbital, and the whole brain was removed, and

snap frozen using liquid nitrogen. For the naïve brain, mice were euthanized and

subsequently brains were removed and flash-frozen in liquid nitrogen. The brains were

then stored at -80 °C until further use.

The brain samples were homogenized to a fine powder using a mortar and pestle

set in a dry ice bath. The fine brain powder was then transferred to pre-chilled micro

centrifuge tubes (Eppendrof, Hamburg, Germany). Powdered brain samples were

stored at -80°C until further use. The brain powder was then lysed with 1% Triton X-100

lysis buffer (Sigma Aldrich, St. Louis, MO) containing 20 mM Tris-HCl (Fisher Scientific,

Fair Lawn, New Jersey) pH 7.0, 5 mM of ethylenediamidetetraacetic acid (EDTA)

(Fisher Scientific, Fair Lawn, New Jersey), and 1M dithiotheitol (DTT) (Sigma Aldrich,

St. Louis, MO) (final concentration in the solution is 1 mM) in double distilled water. The

brain samples were then incubated in the lysis buffer for 90-120 minutes at 4°C kept on

a tube revolver (Thermo Scientific) at a low speed. Following the incubation, the

33

samples were centrifuged at 14,500 rpm for 15 minutes at 4°C, and the supernatant

were transferred to new tubes. The protein concentration was determined by performing

protein assay using a DC protein assay (Bio Rad, Hercules, CA).

Digestion of Proteins

Naïve brain lysate equivalent to 110 μg of protein concentration was used per

sample. 5 μL of 200 mM dithiotreitol (DTT) was added to each sample for reduction

followed by 5 μL of 200 mM calcium chloride (Acros Organics, Geel, Belgium) solution

for activation of protease calpain. Water was added to a final volume of 100 μL. Finally,

protease calpain-1 (Calbiochem, Tullagreen, Larrighwohill, Co. Cork, Ireland) was

added in the ratio of 1:120 (enzyme to protein) and incubated for one hour at room

temperature. The naïve brain lysate was incubated with only calcium chloride solution

for 4 hours at room temperature. Following incubation, calpain inhibitor IV (Calbiochem,

Tullagreen, Larrighwohill, Co. Cork, Ireland) was added to inhibit further digestion by

calpain-1.

The digested samples were filtered through a 10,000 Da molecular weight cut-off

(MWCO) membrane filter (Sartorius Stedim Biotech, Goettingen, Germany). This

filtration technique aids in filtering molecules that are smaller than or equal to 10,000

Da. The filtrate was then concentrated using speed vac (Thermo Scientific) to a volume

of 5 µL. The samples were then reconstituted with water with 0.1% formic acid. These

samples were run by LC-MS/MS on Thermo Scientific LTQ XL with Waters NanoAcquity

UPLC system.

The molecules that were 10,000 Da and higher were retained in the top part of

the filter. This concentrate was analyzed by the western blot. The samples for western

blot were mixed with 8X sample buffer in the ratio of 1:1. The proteins from the samples

34

were separated by gel electrophoresis. The samples were loaded on 18% tris-glycine

mini protein gel (Invitrogen, Life Technologies). The samples were allowed to run for 60

min at a constant voltage of 200V. After separation, the proteins were transferred from

the gel to a PVDF membrane using iBlot transfer (Invitrogen, Life Technologies, Van

Allen Way Carlsbad, CA). Following the transfer, the PVDF membrane was blocked by

adding the blot to a 5% non-fat milk solution prepared in TBST for one hour. After

blocking, the membrane was incubated overnight at 4°C with continuous shaking with

primary antibodies. Monoclonal anti-mouse fodrin for alpha-spectrin, polyclonal anti-

rabbit glial fibrillary acidic protein (GFAP) and monoclonal anti-mouse myelin basic

protein (MBP) were used with a dilution factor of 1:1000 prepared in 5% milk. Polyclonal

anti-rabbit neurogranin was used with a dilution factor of 1:500 prepared in 5% milk. The

following day the membrane was washed three times with TBST. Then the membrane

was probed with alkaline phosphatase-conjugate goat and rabbit secondary antibody

with a dilution of 1:1000 in 5% milk for one hour at room temperature. The membrane

was again washed three times with TBST. Finally, immunoreactivity was detected by

using 5-bromo-4-chloro-indolylphosphate (BCIP) nitroblue tetrazolium phosphate

substrate. The substrate gave the probed proteins a purple color. The membrane was

then washed with water and dried before it was scanned.

Instrumentation

NanoAcquity UPLC (Waters, Milford, MA) was used for separation of proteins by

reversed phase chromatography. 3 μL of each sample was loaded onto a 5 µm

Symmetry 180 µm x 20 mm trapping column followed by separation on a BEH 130 C18

100 μm x 100 mm, 1.7 μm particle size analytical column at 300 nL/min. Mobile phases

used consisted of solvent A as water with 0.1% formic acid and solvent B as acetonitrile

35

with 0.1% formic acid, purchased from Honeywell, Muskegon, MI. Each sample

separation was achieved over a run time of 115 min. The mobile phase gradient used

over the 115 min run time was 1% to 40 % of solvent B for 90 min, followed by 40% to

100% of solvent B for 10 min and held 5 min before returning to the initial composition

of 1% solvent B. Tandem mass spectrometry was performed on a LTQ XL (Thermo,

San Jose, California) using data dependent acquisition method to select the top 10 most

abundant peaks using Xcalibur 2.0.7 (Thermo). The analysis was set up for a full scan

recorded between m/z 350 – 2000, and a MS/MS scan to generate product ion spectra

to determine amino acid sequence in consecutive instrument scans of the ten most

abundant peaks in the spectrum. The method was set to perform full-MS and

subsequent MS/MS scan on the top 10 most intense ions from the full MS scan

spectrum with dynamic exclusion. The dynamic exclusion puts a mass in the exclusion

list once the MS/MS is acquired and subsequently performs MS/MS on the second most

intense ion. Dynamic exclusion was enabled with a repeat count of 2 within 30 seconds,

a mass list size of 50, an exclusion duration 30 seconds, the low mass width was 0.5

and the high mass width was 1.5.

The MS/MS data was analyzed using Proteome Discoverer 1.3 (Thermo) to

search against Uniprot mouse database version 2014_09 (51,196 sequences and

24,539,856 residues) using SEQUEST algorithm, with no enzyme. Unlike trypsin that

has a known cleavage site; at the carboxyl side of the amino acids lysine or arginine,

calpain do not have a known cleavage site. Because of this reason the data was

searched with no enzyme as option. The search was achieved using the average mass

for matching the precursor with a fragment ion mass tolerance of 0.8 Da and a

36

precursor ion tolerance of 0.8 Da. Carbamidomethylation of cysteine was selected as a

static modification, while the oxidation of methionine was selected as a dynamic

modification. Two missed cleavages for the enzyme were permitted.

Results and Discussion

To study the breakdown of protein following an injury, in vitro digestion using

calpain as protease was performed. Calpain activation takes place by hyperactivation

under pathological conditions involving increased concentration of calcium in the cell

due to neuronal injury. Thus, calpain cleavage products may provide useful biomarkers

for the presence of neuronal injury. Therefore by performing in vitro digestion, protein

breakdown products were identified based on the mass spectrometry data and then

validated by western blot.

A schematic of workflow is shown in Figure 2-2. The digestion of brain lysate by

calpain produced smaller peptides. Filtering the digested lysate through the 10,000 Da

MWCO membrane filter was performed to separate the smaller fragmented peptides

and proteins that were then analyzed by mass spectrometry. The larger peptides and

proteins that were retained on the filter were analyzed by western blot. Western blot

served as a validation technique. For western blot, the antibodies selected were based

on the mass spectrometry data, antibody availability, and based on the other

experiments performed in parallel in the lab.

Mass spectrometry

MS/MS-based proteomics studies are based on peptides. In tandem mass

spectrometry (MS/MS), a precursor ion corresponding to a peptide was selected in MS

for further fragmentation in MS/MS. The resulting fragmentation spectra were then

compared to fragmentation spectra in a database, using software Proteome Discoverer.

37

All MS/MS data was collected on the LTQ XL. Since there is no specific cleavage site

for calpain proteolysis, the MS/MS data was searched using no enzyme against mouse

database.

From the database search results, the proteins were selected based on the hit

score. Proteins with a score of 1.5 and higher were selected. The protein score is the

sum of all peptide cross-correlation (Xcorr) values, where the number of fragment ions

is scored that are common to two different peptides with the same precursor mass and

calculates the cross-correlation score for all candidate peptides queried from the

database. About 593 proteins were identified in the undigested cortex lysate (control),

approximately 1005 proteins in the cortex lysate incubated with only calcium chloride

and about 1869 proteins in the cortex lysate digested with calpain. Similarly, in the

undigested corpus callosum (control) about 733 proteins were identified, 682 proteins in

the calcium chloride only incubated lysate and approximately 1720 proteins were

identified in the calpain digested corpus callosum lysate. Lastly, 520 proteins in the

undigested hippocampus lysate, 765 proteins in the calcium only incubated lysate and

1715 proteins in the calpain digested hippocampus lysate were identified. There were a

few proteins found common in calcium only and calpain-digested samples in each brain

region lysates. A Venn diagram showing this overlap is shown in Figure 2-3. A list of

possible protein biomarkers, shown in Table 2-1, was identified from in vitro digested

naïve brain lysate based on the search score, sequence coverage, relevance to brain

diseases and some previous experiments done in the lab. From this possible biomarker

list, proteins with high score and low molecular weight like neurogranin, myelin basic

protein (MBP), and brain soluble acidic protein-1 were selected. Peptides identified from

38

the database search for these proteins were mapped in Figure 2-4, 2-8, 2-12 for these

proteins. MS/MS spectra for these peptides were obtained. Neurogranin is a low

molecular weight protein with 78 amino acids. Figure 2-4 shows the mouse neurogranin

sequence with identified peptides from the database search results. The MS/MS data

show the fragmentation of peptides; –AAKIQASF (m/z 419) (Figure 2-5), –

AAAAKIQASF (m/z 490) (Figure 2-6) and –GPGPGGPGGAGGARGGAGGGPSGD

(m/z 889) (Figure 2-7). The fragment ions for identified for these peptides from the

database are mapped in respective Tables. Mouse BASP-1 is a 22-kDa protein. Figure

2-8 shows the BASP-1 sequence with mapped peptides identified from database search

results. The MS/MS spectrum (Figure 2-9) shows the fragmentation of the peptide –

EAPAAAASSEQSV (m/z 610). Figure 2-10 and 2-11 displays the identified product ions

for the other two BASP1 peptides –EAPAAAASSEQSVAVKE and –

AEPAPSSKETPAASEAPSS respectively. Similarly, MBP peptides were also mapped.

Figure 2-8 shows mouse MBP isoform with two peptides identified from the database

search. The MS/MS spectra (Figure 2-9) show fragmentation for MBP peptide –

KNIVTPRTPPP and the identified fragment ions for this peptide. The fragment ions

identified for the other MBP peptide, - LATASTMDHAR are shown in Figure 2-14.

The CCI samples were processed similar to the in vitro digested samples. From

the database search results, the proteins were selected based on the hit score. For the

CCI samples, the selection criterion for the proteins was same as the in vitro digestion

samples, that is, proteins with a score of 1.5 and higher were selected. In the CCI

mouse brain samples, there were 345 hits were unique to control cortex lysate

compared to 2380 hits for the CCI cortex lysate. The corpus callosum region lysate

39

showed 396 hits for the control and 2735 protein hits for CCI lysate. The hippocampus

region lysate showed 304 hits for the control and 2723 hits for CCI lysate. A Venn

diagram showing the number hits unique and common to each region is shown in

Figure 2-15. A list of possible protein biomarkers from the CCI brain lysates was

prepared and was compared to the list of proteins identified by in vitro digestion. There

were a few proteins that were common as observed by in vitro digestion. A list of

potential protein biomarkers observed in CCI samples is shown in Table 2-2. This list

was based on the search score, sequence coverage, proteins identified by in vitro

digestion, relevance to brain diseases and some previous experiments done in the lab.

Neurogranin and BASP-1, which were identified by in vitro digestion, were not

observed in the CCI samples by mass spectrometry. But neurogranin was identified by

western blot in all the CCI samples. MBP and its fragments were observed in the CCI

samples, especially in the corpus callosum region brain lysates.

Western Blot

A western blot was performed on the calpain digested naïve brain lysates and

CCI brain lysates of different brain regions (cortex, corpus callosum and hippocampus)

that were retained on the 10,000 Da MWCO filter after the filtration of the lysates. The

proteins probed were alpha-spectrin, GFAP, Neurogranin, and MBP. Western blot was

used as a confirmation or validation technique for some proteins that were identified by

mass spectrometry.

In the digested brain lysate samples (from all three brain regions – cortex, corpus

callosum, and hippocampus) showed increase in breakdown products of spectrin,

SBDP 150 and SBDP120 (Figure 2-16A, 2-17A, and 2-18A). There were some spectrin

breakdown products present endogenously in the control lysate but the levels were low.

40

The intact and breakdown products were quantified based on color density of the bands

(Figure 2-16B, 2-17B, and 2-18B). The levels of spectrin breakdown products were

increased in all the digested samples. These results show that spectrin was subjected

to proteolysis when incubated with calcium and calpain.

In the digested brain lysate samples, from all the three brain regions, the GFAP

signal was either low or was inconsistent. Figure 2-19 displays a western blot probed

with GFAP antibody. In the corpus callosum and hippocampus regions, GFAP showed

very low signal that made it difficult for quantification. The antibody used for MBP

probed the MBP breakdown products.

The MBP breakdown product was observed in all the digested samples (Figure

2-20 A). It was also observed in the control samples but after quantifying the color

density, the level was low compared to the calpain-digested samples (Figure 2-20 B, C,

D). In the calcium-only digested samples the level of MBP breakdown product was low

compared to the control.

The novel protein biomarker, neurogranin displayed distinct increase in the

breakdown products in the calpain digested samples in all the three brain region

lysates. Figure 2-21 A, 2-22 A, 2-23 A, show the western blot for digested brain lysate

samples probed with anti-neurogranin. The intact and breakdown products of

neurogranin were also quantified based on the color density of the bands (Figure 2-21

B, 2-22 B, 2-23 B). This shows that neurogranin is subjected to proteolysis following

calpain digestion.

Following the in vitro digestion study, the CCI samples were processed to study

the effect of injury on these proteins. Western blot was performed on these samples that

41

were retained on the 10-kDa MWCO membrane filter. Figure 2-24A display western blot

of CCI brain lysates of all the three brain regions probed with alpha spectrin antibody.

The breakdown products showed an increase in the CCI samples (Figure 2-24B) when

quantified based on the color density of the bands.

In the CCI samples, GFAP and its breakdown products were detected but the

signal was very low in the control samples. A western blot probed with GFAP antibody

is shown in Figure 2-25. GFAP and its breakdown products were mainly observed in the

corpus callosum and hippocampus region brain lysate. MBP breakdown product

antibody was used to probe the blot. The MBP breakdown products were observed in

the CCI samples at 18 kDa. Figure 2-26A displays the blot probed with MBP breakdown

product and Figure 2-26 B-D shows the quantified data for the breakdown product of

MBP.

The blot was also probed with neurogranin antibody (Figure 2-27A). The

breakdown product that was seen distinctly in the digested samples was seen in the

CCI samples. But after quantifying the intact band for neurogranin (Figure 2-27B) in the

CCI samples, it shows decrease in all the CCI samples when compared to the intact

neurogranin in the control sample. This shows that there is less fragmentation of

neurogranin following injury. Neurogranin was not identified by mass spectrometry but

its breakdown was confirmed by western blot.

Conclusions

In vitro digestion using calpain as a protease served as a model for calpain

activated breakdown of proteins following brain injury. In this study, potential protein

biomarkers were identified that are subjected to degradation by calpain activation due to

increased concentration of calcium ions in the cytoplasm following injury to the brain. In

42

identifying the potential biomarkers, naïve brain lysate was digested with the protease

calpain in presence of calcium. By filtering the digested lysate through the 10-kDa

membrane filter, the proteolytic peptides smaller than 10-kDa were separated. These

smaller proteolytic peptides were analyzed by reversed phased liquid chromatography

with tandem mass spectrometry (LC-MS/MS). The MS/MS data was searched using a

database called Proteome Discoverer. The high molecular weight peptides and proteins

that were retained on the filter were analyzed using western blot by probing the blot with

few specific antibodies that were selected based on the mass spectrometry data,

antibody availability, and on the other experiments performed in parallel in the lab.

Based on the database search results, proteins with a score of 1.5 were selected. From

these list some brain specific potential biomarkers were selected. The CCI mouse brain

samples were processed similar to the naïve brain lysate, except for the addition of

calcium and calpain in the CCI samples. The list of proteins identified in the CCI

samples matched the list of proteins identified by in vitro digestion of naïve brain lysate.

Western blot technique was an added advantage that confirmed some proteins

identified by mass spectrometry. Neurogranin, which was not observed in the CCI

samples, was confirmed by western blot.

Thus mass spectrometry serves a better platform for biomarker study when used

with proper bioinformatics tools. Western blot also provided an added benefit of

confirmation to identify few protein of interest.

43

Figure 2-1. Calpain and excitotoxicity. Excessive glutamate levels during cerebral ischemia causing overactivation of ionotropic receptors allowing Ca2+/Na+ influx. Elevated free intracellular Na+ levels trigger opening of the Ca2+ channels, resulting in elevated intracellular Ca2+ levels and leading to activation of Ca2+-dependent systems. (Adapted from K.K.W.Wang; et al) [47]

44

Figure 2-2. Workflow of sample processing of mouse brain lysates (control and CCI).

Proteolysis (TBI, CCI, or

Ca2+

/Calpain)

LC-MS/MS Western Blot

10-kDa MWCO

filter

Proteolytic peptides ≤ 10-kDa

> 10-kDa

Database search

Biomarker Identification

Spin

45

Figure 2-3. Venn diagram showing the number of proteins identified using Proteome

Discoverer based on the LC-MS/MS data from digested naïve brain lysate of cortex (A), corpus callosum (B) and hippocampus (C) region. In vitro digestion was performed with the naïve brain lysate of the cortex, hippocampus and corpus callosum region using two conditions. The first condition involves incubating with calcium chloride only and the second condition involves incubating with calpain-1 along with calcium chloride. The data are based on the filtrate from filtration through the 10,000 Da MWCO filter.

Control593

Calpain1869

Calcium1005

Control 733

Calpain1720

Calcium682

Control520

Calpain1715

Calcium765

142 86

217

42 86 160

168

35 32 73

200

124

Cortex

A Corpus

Callosum

Hippocampus B C

46

Table 2-1. List of protein biomarkers identified by performing in vitro digestion of mouse brain lysate using LC-MS/MS.

Accession Number

Protein name Gene name Molecular weight (kDa)

H7BX08 Calmodulin regulated spectrin-associated protein 2 CAMSAP2 166.0

E9Q5B0 Calmodulin regulated spectrin-associated protein 3 CAMSAP3 135.2

Q8VHY0 Chondroitin sulfate proteoglycan 4 CSPG4 252.2

Q04447 Creatine kinase B-type Ckb 42.7

P03995 Glial fibrillary acidic protein GFAP 52 P48318 Glutamate decarboxylase 1 GAD1 66.6 G3X9H5 Huntingtin Htt 344.6 A2ARP8 Microtubule-associated protein 1A MAP1A 325.7

P14873 Microtubule-associated protein 1B MAP1B 270.1

P20357 Microtubule-associate protein 2 MAP2 199.0 Q7TSJ2 Microtubule-associated protein 6 MAP6 96.4

Q9R1L5 Microtubule-associated serine/threonine-protein kinase 1 MAST1 170.9 P04370 Myelin basic protein MBP 27.2 E0CY11 Neurexin-1 NRXN1 164.1 Q6P9K9 Neurexin-3 NRXN3 173.3 P55066 Neurocan NCAN 137.1 Q810U3 Neurofascin NFASC 137.9 P19246 Neurofilament-H NEFH 116.9 P08551 Neurofilament-L NEFL 61.5

P08553 Neurofilament-M NEFM 95.8

P60761 Neurogranin NRGN 7.5

P08032 Spectrin alpha chain, erythrocytic 1 SPTA1 279.7

Q62261 Spectrin, beta, nonerythrocytic 1 SPTBN1 274.1

Q64332 Synapsin II SYN2 63.3

Q91ZZ3 Synuclein, beta SNCB 14.0

47

1 MDCCTESACS KPDDDILDIP LDDPGANAAAAKIQASFRGH MARKKIKSGE

51 CGRKGPGPGGPGGAGGARGGAGGGPSGD

Figure 2-4. Mouse neurogranin sequence with three mapped peptides, identified by

database search result.

A

B

b⁺ b²⁺ Seq. y⁺ y²⁺

1 72.08609 36.54674 A 8

2 143.16479 72.08609 A 764.90049 382.95394 7

3 271.33929 136.17334 K 693.82179 347.41459 6

4 384.49909 192.75324 I 565.64729 283.32734 5

5 512.63000 256.81870 Q 452.48749 226.74744 4

6 583.70870 292.35805 A 324.35658 162.68199 3

7 670.78670 335.89705 S 253.27788 127.14264 2

8 F 166.19988 83.60364 1

Figure 2-5. MS/MS spectra for the neurogranin peptide AAKIQASF. (A).MS/MS spectra

displaying the fragment ions for this peptide. (B). Identified all b- and y-type ions for the neurogranin peptide AAKIQASF shown in red and blue from the database search results.

C:\XCalibur\...\CC-Triton-Ca+Calpain 3/29/2015 8:18:38 PM

RT: 0.00 - 115.01

0 10 20 30 40 50 60 70 80 90 100 110

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

34.72 36.76

32.3638.63

48.20

45.79

43.25

29.59 49.14

51.06

64.3525.68

51.8764.82

107.9821.99 73.4652.032.32 20.83 63.87

11.46 65.2952.66 98.69 99.7655.06 98.0974.14 108.1587.9279.8410.72 14.82101.90

NL:

3.56E6

Base Peak

MS CC-Triton-

Ca+Calpain

CC-Triton-Ca+Calpain #1600 RT: 26.19 AV: 1 NL: 1.98E4F: ITMS + c ESI d Full ms2 418.73@cid35.00 [105.00-850.00]

150 200 250 300 350 400 450 500 550 600 650 700 750 800

m/z

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

409.83

670.90

512.69

693.96

166.23583.80

324.45253.37

565.80

336.04 400.76452.61129.19 652.91495.68271.40

764.95306.50 528.77379.90235.35 434.67207.38 621.89 705.87 785.28

y1

y2 y3

y5

y6

y7 b3

b5

b6

b7 AAKIQASF

48

b⁺ b²⁺ Seq. y⁺ y²⁺

1 72.08609 36.54674 A 10

2 143.16479 72.08609 A 907.05789 454.03264 9

3 214.24349 107.62544 A 835.97919 418.49329 8

4 285.32219 143.16479 A 764.90049 382.95394 7

5 413.49669 207.25204 K 693.82179 347.41459 6

6 526.65649 263.83194 I 565.64729 283.32734 5

7 654.78740 327.89740 Q 452.48749 226.74744 4

8 725.86610 363.43675 A 324.35658 162.68199 3

9 812.94410 406.97575 S 253.27788 127.14264 2

10 F 166.19988 83.60364 1

Figure 2-6. Identified b- and y-type ions for the neurogranin peptide AAAAKIQASF

shown in red and blue.

49

#1 b⁺ b²⁺ Seq. y⁺ y²⁺ #2

1 58.05909 29.53324 G 24

2 155.17589 78.09164 P 1720.75258 860.87999 23

3 212.22759 106.61749 G 1623.63578 812.32159 22

4 309.34439 155.17589 P 1566.58408 783.79574 21

5 366.39609 183.70174 G 1469.46728 735.23734 20

6 423.44779 212.22759 G 1412.41558 706.71149 19

7 520.56459 260.78599 P 1355.36388 678.18564 18

8 577.61629 289.31184 G 1258.24708 629.62724 17

9 634.66799 317.83769 G 1201.19538 601.10139 16

10 705.74669 353.37704 A 1144.14368 572.57554 15

11 762.79839 381.90289 G 1073.06498 537.03619 14

12 819.85009 410.42874 G 1016.01328 508.51034 13

13 890.92879 445.96809 A 958.96158 479.98449 12

14 1047.11679 524.06209 R 887.88288 444.44514 11

15 1104.16849 552.58794 G 731.69488 366.35114 10

16 1161.22019 581.11379 G 674.64318 337.82529 9

17 1232.29889 616.65314 A 617.59148 309.29944 8

18 1289.35059 645.17899 G 546.51278 273.76009 7

19 1346.40229 673.70484 G 489.46108 245.23424 6

20 1403.45399 702.23069 G 432.40938 216.70839 5

21 1500.57079 750.78909 P 375.35768 188.18254 4

22 1587.64879 794.32809 S 278.24088 139.62414 3

23 1644.70049 822.85394 G 191.16288 96.08514 2

24 D 134.11118 67.55929 1

Figure 2-7. Identified b- and y-type ions for the neurogranin peptide

GPGPGGPGGAGGARGGAGGGPSGD shown in red and blue.

50

1 MGGKLSKKKK GYNVNDEKAK DKDKKAEGAG TEEEGTPKES EPQAAADATE VKESTEEKPK

61 DAADGEAKAE EKEADKAAAA KEEAPKAEPE KSEGAAEEQP EPAPAPEQEA AAPGPAAGGE

121 APKAGEASAE STGAADGAAP EEGEAKKTEAP AAAGPEAKS DAAPAASDSK PSSAEPAPSS

181 KETPAASEAPSS AAKAPAPA APAAAEPQA EAPAAAASSEQSVAVKE

Figure 2-8. Mouse BASP-1 sequence with two mapped peptides identified by database

search result.

51

A

B

b⁺ b²⁺ Seq. y⁺ y²⁺

1 130.12299 65.56519 E 13

2 201.20169 101.10454 A 1089.14629 545.07684 12

3 298.31849 149.66294 P 1018.06759 509.53749 11

4 369.39719 185.20229 A 920.95079 460.97909 10

5 440.47589 220.74164 A 849.87209 425.43974 9

6 511.55459 256.28099 A 778.79339 389.90039 8

7 582.63329 291.82034 A 707.71469 354.36104 7

8 669.71129 335.35934 S 636.63599 318.82169 6

9 756.78929 378.89834 S 549.55799 275.28269 5

10 885.90489 443.45614 E 462.47999 231.74369 4

11 1014.03580 507.52160 Q 333.36439 167.18589 3

12 1101.11380 551.06060 S 205.23348 103.12044 2

13 V 118.15548 59.58144 1

Figure 2-9. MS/MS spectra for the BASP-1 peptide EAPAAAASSEQSV. (A) MS/MS

spectra displaying the fragment ions for this peptide. (B). Identified b- and y-type ions for the BASP1 peptide EAPAAAASSEQSV shown in red and blue from the database search results

C:\XCalibur\...\Hippo-Triton-Ca+Calpain 3/30/2015 9:05:58 AM

RT: 0.00 - 115.07

0 10 20 30 40 50 60 70 80 90 100 110

Time (min)

0

10

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30

40

50

60

70

80

90

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Re

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Ab

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44.61

44.44

46.23

44.10 107.94

46.54

37.14

35.6532.69

10.85

42.26 46.8611.01 26.9738.51

47.207.2399.10

21.012.43 98.43 99.6048.02 108.2913.56 100.5895.7488.7986.6354.0318.81 83.14 109.4266.3358.22 79.0967.076.14 102.17

NL:

2.82E6

Base Peak

MS Hippo-

Triton-Ca+Calpain

Hippo-Triton-Ca+Calpain #1079 RT: 16.65 AV: 1 NL: 7.77E2F: ITMS + c ESI d Full ms2 610.01@cid35.00 [155.00-1235.00]

300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

601.02

582.80

636.87

707.89511.76

778.96

669.88549.73

850.10 1014.12440.68696.61

756.93 823.27886.13

333.60 542.21462.70422.66369.60 1101.13741.43 798.36 940.35323.55 1000.25381.86 1071.79894.61

b4

b5

b6

b7 y6

b8

y7

b9

y8

y9 b10

b11

b12

EAPAAAASSEQSV

52

b⁺ b²⁺ Seq. y⁺ y²⁺

1 130.12299 65.56519 E 17

2 201.20169 101.10454 A 1516.64789 758.82764 16

3 298.31849 149.66294 P 1445.56919 723.28829 15

4 369.39719 185.20229 A 1348.45239 674.72989 14

5 440.47589 220.74164 A 1277.37369 639.19054 13

6 511.55459 256.28099 A 1206.29499 603.65119 12

7 582.63329 291.82034 A 1135.21629 568.11184 11

8 669.71129 335.35934 S 1064.13759 532.57249 10

9 756.78929 378.89834 S 977.05959 489.03349 9

10 885.90489 443.45614 E 889.98159 445.49449 8

11 1014.03580 507.52160 Q 760.86599 380.93669 7

12 1101.11380 551.06060 S 632.73508 316.87124 6

13 1200.24660 600.62700 V 545.65708 273.33224 5

14 1271.32530 636.16635 A 446.52428 223.76584 4

15 1370.45810 685.73275 V 375.44558 188.22649 3

16 1498.63260 749.82000 K 276.31278 138.66009 2

17 E 148.13828 74.57284 1

Figure 2-10. Identified b- and y-type ions for the BASP1 peptide

EAPAAAASSEQSVAVKE shown in red and blue.

53

b⁺ b²⁺ Seq. y⁺ y²⁺

1 72.08609 36.54674 A 19

2 201.20169 101.10454 E 1743.82108 872.41424 18

3 298.31849 149.66294 P 1614.70548 807.85644 17

4 369.39719 185.20229 A 1517.58868 759.29804 16

5 466.51399 233.76069 P 1446.50998 723.75869 15

6 553.59199 277.29969 S 1349.39318 675.20029 14

7 640.66999 320.83869 S 1262.31518 631.66129 13

8 768.84449 384.92594 K 1175.23718 588.12229 12

9 897.96009 449.48374 E 1047.06268 524.03504 11

10 999.06519 500.03629 T 917.94708 459.47724 10

11 1096.18199 548.59469 P 816.84198 408.92469 9

12 1167.26069 584.13404 A 719.72518 360.36629 8

13 1238.33939 619.67339 A 648.64648 324.82694 7

14 1325.41739 663.21239 S 577.56778 289.28759 6

15 1454.53299 727.77019 E 490.48978 245.74859 5

16 1525.61169 763.30954 A 361.37418 181.19079 4

17 1622.72849 811.86794 P 290.29548 145.65144 3

18 1709.80649 855.40694 S 193.17868 97.09304 2

19 S 106.10068 53.55404 1

Figure 2-11. Identified b- and y-type ions for the BASP1 peptide

AEPAPSSKETPAASEAPSS shown in red and blue.

54

1 MGNHSGKREL SAEKASKDGE IHRGEAGKKR SVGKLSQTAS EDSDVFGEAD

51 AIQNNGTSAE DTAVTDSKHT ADPKNNWQGA HPADPGNRPH LIRLFSRDAP

101 GREDNTFKDR PSESDELQTI QEDPTAASGG LDVMASQKRP SQRSKYLATA

151 STMDHARHGF LPRHRDTGIL DSIGRFFSGD RGAPKRGSGK DSHTRTTHYG

201 SLPQKSQHGR TQDENPVVHF FKNIVTPRTPPPSQGKGGRD SRSGSPMARR

Figure 2-12. Mouse MBP isoform sequence with two mapped peptides identified by

database search result. One is marked in red and the second one is underlined.

55

A

B

b⁺ b²⁺ Seq. y⁺ y²⁺

1 129.18189 65.09464 K 11

2 243.28579 122.14659 N 1092.28458 546.64599 10

3 356.44559 178.72649 I 978.18068 489.59404 9

4 455.57839 228.29289 V 865.02088 433.01414 8

5 556.68349 278.84544 T 765.88808 383.44774 7

6 653.80029 327.40384 P 664.78298 332.89519 6

7 809.98829 405.49784 R 567.66618 284.33679 5

8 911.09339 456.05039 T 411.47818 206.24279 4

9 1008.21019 504.60879 P 310.37308 155.69024 3

10 1105.32699 553.16719 P 213.25628 107.13184 2

11 P 116.13948 58.57344 1

Figure 2-13. MS/MS spectra for the MBP peptide KNIVTPRTPPP. (A) MS/MS spectra

displaying the fragment ions for this peptide. (B). Identified b- and y-type ions for the MBP peptide KNIVTPRTPPP shown in red and blue from the database search results.

C:\XCalibur\...\CCI-409-CC 5/3/2015 11:23:19 PM

RT: 0.00 - 115.01

0 10 20 30 40 50 60 70 80 90 100 110

Time (min)

0

10

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40

50

60

70

80

90

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Re

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Ab

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48.71

49.6347.66

46.6533.1530.72

45.5127.9451.55

35.2124.41

37.0744.33

38.72 52.3140.01

19.13

53.08 99.1519.52 53.84

54.74 100.52

57.2218.93 91.53 95.27 101.3080.7262.40 71.76 90.9676.5114.9611.23 107.9810.022.58

NL:

5.61E7

Base Peak

MS CCI-409-

CC

CCI-409-CC #935 RT: 15.22 AV: 1 NL: 4.97E2F: ITMS + c ESI d Full ms2 611.12@cid35.00 [155.00-1235.00]

200 300 400 500 600 700 800 900 1000 1100 1200

m/z

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

602.54

765.95

664.88 865.02

1092.21

911.07355.29978.14556.82

525.70

310.51 455.68

438.70 509.72226.23965.22 1103.26783.88285.34 722.03428.43 611.29 1076.36847.45 1008.17 1161.93

KNIVTPRTPPP

b4 y3

y6

b5

y7

y8

b8 y9

y10

56

b⁺ b²⁺ Seq. y⁺ y²⁺

1 114.16719 57.58729 L 11

2 185.24589 93.12664 A 1061.16428 531.08584 10

3 286.35099 143.67919 T 990.08558 495.54649 9

4 357.42969 179.21854 A 888.98048 444.99394 8

5 444.50769 222.75754 S 817.90178 409.45459 7

6 545.61279 273.31009 T 730.82378 365.91559 6

7 676.81219 338.90979 M 629.71868 315.36304 5

8 791.90069 396.45404 D 498.51928 249.76334 4

9 929.04209 465.02474 H 383.43078 192.21909 3

10 1000.12079 500.56409 A 246.28938 123.64839 2

11 R 175.21068 88.10904 1

Figure 2-14. Identified b- and y-type ions for the MBP peptide LATASTMDHAR shown

in red and blue.

57

Figure 2-15. Venn diagram showing the number of proteins identified using Proteome Discoverer based on the LC-MS/MS data from CCI brain lysate samples of cortex (A), corpus callosum (B) and hippocampus (C) region. These data are based on the filtrate from filtration through the 10,000 Da MWCO filter.

Control

345

CCI

2380

Control

396

CCI

2735

Control

304

CCI

2723

Cortex A Hippocampus B C

162 167 129

Corpus Callosum

58

Table 2-2. List of protein biomarkers identified in CCI mouse brain lysate by LC-MS/MS.

Accession number

Full protein Name Gene Name Molecular weight (kDa)

Q8R5A3 Amyloid beta A4 precursor protein-binding family B member 1-interacting protein Apbb1ip 74.3

H7BX08 Calmodulin-regulated spectrin-associated protein 2 Camsap2 166 E9Q5B0 Calmodulin-regulated spectrin-associated protein 3 Camsap3 135.2 Q8VHY0 Chondroitin sulfate proteoglycan 4 Cspg4 252.2 P48318 Glutamate decarboxylase 1 Gad1 66.6 G3X9H5 Huntingtin Htt 344.6 Q9Z0E0-2 Neurochondrin Ncdn 77.4 A2ARP8 Microtubule-associated protein 1A Map1a 325.7 P14873 Microtubule-associated protein 1B Map1b 270.1 P20357 Microtubule-associated protein 2 Map2 199 Q7TSJ2 Microtubule-associated protein 6 Map6 96.4 P04370 Myelin basic protein Mbp 27.2 E0CY11 Neurexin-1 Nrxn1 164.1 Q6P9K9 Neurexin-3 Nrxn3 173.3 P55066 Neurocan core protein Ncan 137.1 Q810U3 Neurofascin Nfasc 132.1 P19246 Neurofilament heavy polypeptide Nefh 116.9 P08551 Neurofilament light polypeptide Nefl 61.5 Q03517 Secretogranin-2 Scg 70.6 P08032 Spectrin alpha chain, erythrocytic 1 Spta1 279.7 A3KGU5 Spectrin alpha chain, non-erythrocytic 1 Sptan1 282.7 Q62261 Spectrin beta chain, non-erythrocytic 1 Sptbn1 274.1 Q64332 Synapsin-2 Syn 63.3 P46097 Synaptotagmin-2 Syt2 47.2 Q7TMM9 Tubulin beta-2A chain Tubb2a 49.9 Q9D6F9 Tubulin beta-4A chain Tubb4a 49.6 P68372 Tubulin beta-4B chain Tubb4b 49.8

59

Figure 2-16. Western blot of the digested naïve brain lysate of cortex region probed with

alpha-spectrin antibody. (A) Western blot displaying the spectrin breakdown products, 150 kDa and 120 kDa observed in calcium and calpain digested samples. (B). Quantitation of the intact, and breakdown products of spectrin. Error bars represent the standard error of the mean (N=3).

A

SBDP (150 kDa)

SBDP (120 kDa)

α-Spectrin

0

20

40

60

80

100

120

140

160

180

Control Ca2+ only Ca+Calpain

Arb

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Un

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Intact

SBDP 150

SBDP 120

B

60

Figure 2-17. Western blot of the digested naïve brain lysate of corpus callosum (CC)

region probed with alpha-spectrin antibody. (A) Western blot displaying the spectrin breakdown products, 150 kDa and 120 kDa observed in calcium and calpain digested samples. (B). Quantitation of the intact, and breakdown products of spectrin. Error bars represent the standard error of the mean (N=3).

SBDP (150/145 kDa)

SBDP (120 kDa)

α-Spectrin

A

0

10

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30

40

50

60

70

80

90

100

Control Ca2+ only Ca+Calpain

Arb

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Intact

SBDP 150

SBDP 120

B

61

Figure 2-18. Western blot of the digested naïve brain lysate of hippocampus region

probed with alpha-spectrin antibody. (A) Western blot displaying the spectrin breakdown products, 150 kDa and 120 kDa observed in calcium and calpain digested samples. (B). Quantitation of the intact, and breakdown products of spectrin. Error bars represent the standard error of the mean (N=3).

SBDP (150/145 kDa)

SBDP (120 kDa)

α-Spectrin A

0

10

20

30

40

50

60

70

80

90

Control Ca2+ only Ca+Calpain

Arb

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Intact

SBDP 150

SBDP 120

B

62

Figure 2-19. Western blot of the digested naïve brain lysate of Cortex region probed

with GFAP antibody displaying the intact and breakdown product (38 kDa).

GFAP- BDP (38 kDa)

GFAP (52 kDa)

63

Figure 2-20. Western blot of the digested naïve brain lysate probed with MBP antibody

(A) Western blot displaying the breakdown product of MBP. (B-D). Quantitation of the breakdown product of MBP. Error bars represent the standard error of the mean (N=3).

MBP (18 kDa)

A

B C

D

0

20

40

60

Control Ca2+ only Ca+Calpain

Arb

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y D

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Un

its Cortex MBP-BDP

0

10

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40

Control Ca2+ only Ca+CalpainArb

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Un

its

Corpus Callosum MBP-BDP

0

20

40

60

Control Ca2+ only Ca+Calpain

Arb

itar

y D

en

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Un

its Hippocampus MBP-BDP

64

Figure 2-21. Western blot of the digested naïve brain lysate of cortex region probed with

neurogranin antibody. (A) Western blot displaying the breakdown product of neurogranin observed in the calcium and calpain digested samples. (B). Quantitation of the intact, and breakdown product of neurogranin. Error bars represent the standard error of the mean (N=3).

Neurogranin

Neurogranin-BDP

A

0

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80

100

120

140

Control Ca2+ only Ca+Calpain

Arb

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Intact

NBDP

B

65

Figure 2-22. Western blot of the digested naïve brain lysate of corpus callosum region

probed with neurogranin antibody. (A) Western blot displaying the breakdown product of neurogranin observed in the calcium and calpain digested samples. (B). Quantitation of the intact, and breakdown product of neurogranin. Error bars represent the standard error of the mean (N=3).

Neurogranin

Neurogranin-

BDP

A

0

20

40

60

80

100

120

140

160

Control Ca2+ only Ca+Calpain

Arb

itar

y D

Ensi

ty U

nit

s

Intact

NBDP

B

66

Figure 2-23. Western blot of the digested naïve brain lysate of hippocampus region

probed with neurogranin antibody. (A) Western blot displaying the breakdown product of neurogranin observed in the calcium and calpain digested samples. (B). Quantitation of the intact, and breakdown product of neurogranin. Error bars represent the standard error of the mean (N=3).

Neurogranin

Neurogranin-BDP

A

0

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60

80

100

120

Control Ca2+ only Ca+Calpain

Arb

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Intact

NBDP

B

67

Figure 2-24. Western blot of CCI brain lysates of all the three regions probed with alpha-

spectrin antibody. (A) Western blot displaying the breakdown product of spectrin observed CCI brain lysate samples. (B-D). Quantitation of the intact and breakdown products of alpha-spectrin. Error bars represent the standard error of the mean (N=3). * Denotes SBDPs (SBDP150 and SBDP120) for the CCI samples is more than the control samples in the corpus callosum (P=0.0207 and P=0.0070 respectively) and hippocampus (P=0.0465 and P=0.0243 respectively) region brain lysates.

SBDP (150 kDa) SBDP (120 kDa)

α-Spectrin

A

0

50

100

150

CortexControl

Cortex-CCI

Arb

itar

y D

en

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Un

its

Intact

SBDP 150

SBDP 120

0

20

40

60

80

CC Control CC-CCI

Arb

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its

Intact

SBDP 150

SBDP 120

0

20

40

60

80

HippoControl

Hippo-CCI 1

Arb

itar

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Un

its

Intact

SBDP 150

SBDP 120

B C

D

* *

* *

68

Figure 2-25. Western blot of CCI brain lysates of all the three regions probed with GFAP

antibody.

GBDP (38 kDa)

GFAP (52 kDa)

A

69

Figure 2-26. Western blot of CCI brain lysates of all the three regions probed with MBP

antibody. (A) Western blot displaying the breakdown product of MBP observed CCI brain lysate samples. (B-D). Quantitation of the breakdown product of MBP. Error bars represent the standard error of the mean (N=3). Error bars represent the standard error of the mean (N=3). * Denotes MBP-BDP for the CCI samples is more than the control samples in the corpus callosum (P=0.0059) and hippocampus (P=0.0321) region brain lysates.

MBP (18 kDa)

A

0

10

20

30

40

Control CCI

Arb

itar

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Un

its

Cortex MBP-BDP

0

20

40

60

Control CCI 1

Arb

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Corpus Callosum MBP-BDP

0

10

20

30

40

50

Control CCI

Arb

itar

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Un

its

Hippocampus MBP-BDP

B C

D

*

*

70

Figure 2-27. Western blot of CCI brain lysates of all the three regions probed with

neurogranin antibody. (A) Western blot displaying the intact neurogranin observed CCI brain lysate samples. (B-D). Quantitation of the intact, and breakdown products of neurogranin. Error bars represent the standard error of the mean (N=3). * Denotes intact neurogranin is reduced in the CCI samples than the control samples which statistically significant for the cortex (P=0.0209) and hippocampus (P=0.0128) samples.

A

Neurogranin

0

20

40

60

80

100

120

Control Cortex-CCI

Arb

itar

y D

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Un

its

Intact

0

50

100

150

200

Control Corpus Callosum-CCI

Arb

itar

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its

Intact

0

20

40

60

80

100

120

Control Hippocampus-CCI

Arb

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Un

its

Intact

B C

D

*

*

71

CHAPTER 3 IDENTIFYING PROTIEN BIOMARKERS IN CLINICAL HUMAN CEREBROSPINAL

FLUID (CSF) SAMPLES

Introduction

Cerebrospinal fluid (CSF) is clear and colorless fluid that surrounds the brain and

spinal cord. The primary function CSF is to provide a cushion, by protecting the brain

and spinal cord from injury. CSF also provides nutrients and chemicals filtered from the

blood to the central nervous system. [48] The removal of waste products from cerebral

metabolism is done via CSF. CSF is obtained by performing a lumbar puncture. In spite

of lumbar puncture being invasive and potentially painful for the patient, CSF is probably

the most informative obtainable fluid for central nervous system (CNS) disease

prognosis. Also, since CSF is in direct contact with the extracellular space of the CNS,

biochemical changes in the brain could potentially be reflected in CSF. [49] Therefore,

CSF constitutes a potential source for clinically useful biomarker candidates of brain

diseases and disorder.

In this work, TBI biomarkers were identified in human CSF samples. The

potential TBI biomarkers identified by in vitro digestion and CCI samples from mouse

brain lysate were extrapolated to human CSF samples. Since CSF carries out removal

of waste products from cerebral metabolism, it would be an ideal biological sample to

study the brain injury biomarkers. Sample preparation similar to CCI brain lysate was

used for the CSF samples.

Experimental

Cerebrospinal fluid (CSF) samples

CSF from subjects suffering TBI was collected after consenting patients

presenting to the emergency department of Ben Taub general hospital, Baylor College

72

of Medicine, Houston, Texas. CSF was collected by trained hospital employees using

buretrol through the CSF drainage system. About 1 mL of the collected CSF was

centrifuged to remove any loose cells and debris. The supernatent was then snap-

frozen and stored at -80°C. In this study, CSF samples (n=12) collected after 24 hours

of injury were used. The control CSF samples (n=6) were purchased from

Bioreclamation Inc.

Sample preparation

The control CSF samples were pooled into 2 sets (3 subjects per set). A volume

of about 250 μL of CSF from pooled control and TBI were used for this study. The CSF

samples were filtered though 10,000 Da molecular weight cut off membrane filter

(Sartorius Stedim Biotech, Goettingen, Germany) by centrifuging the samples at 10,000

rpm for 15 min. The filtrate was then concentrated using a speed vac (Thermo,

Asheville, NC) to a volume of about 5 μL. The samples were then reconstituted in water

with 0.1% formic acid. The reconstituted samples were analyzed by LC-MS/MS.

The part of the sample that was retained on the membrane filter was analyzed by

western blot. The samples were mixed with equal volume of 8X sample buffer before

performing gel electrophoresis. The samples were loaded on 18% tris glycine gel

(Invitrogen, Life Technologies, Allen Way Carlsbad, CA) and allowed to run for 60 min

at a constant voltage of 200V. Following the separation of proteins by electrophoresis,

they were transferred from the gel to a solid support, a PVDF membrane using iBlot

transfer (Invitrogen, Life Technologies). After the transfer, the membrane was blocked

by incubating in 5% non-fat milk prepared in TBST for 1 hr. Then the membrane was

incubated with primary antibodies at 4°C with continuous shaking, overnight. The

primary antibodies, monoclonal anti-mouse fodrin for alpha-spectrin, polyclonal anti-

73

rabbit glial fibrillary acidic protein (GFAP) and monoclonal anti-mouse myelin basic

protein (MBP) were used with a dilution factor of 1:1000 and polyclonal rabbit anti-

neurogranin with a dilution factor of 1:500 prepared in 5% non-fat milk. The next day,

the membrane was washed with TBST 3 times. Then the membrane was probed with

alkaline phosphatase-conjugate goat and rabbit secondary antibody with a dilution of

1:1000 prepared in 5% milk for one hour at room temperature. The membrane was then

washed with TBST 3 times. Lastly, the immunoreactivity was detected by using 5-

bromo-4-chloro-indolylphosphate (BCIP) nitroblue tetrazolium phosphate substrate. The

substrate gave a purple color to the probed proteins. Once developed, the membrane

was washed with water to stop the activity and air dried before it was scanned.

Instrumentation

The samples to be analyzed by LC-MS/MS were separated on NanoAcquity

UPLC (Waters, Milford, MA). 3 μL of each sample was loaded onto a trapping column (5

µm Symmetry 180 µm x 20 mm) followed by separation on a BEH 130 C18 100 μm x

100 mm, 1.7 μm particle size, analytical column at 300 nL/min. Water with 0.1% formic

acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B) were used as mobile

phases. The run time over which separation was achieved was 115 min. The mobile

phase gradient used over the 115 min run time was 1% to 40 % of solvent B for 90 min,

followed by 40% to 100% of solvent B for 10 min and held 5 min before returning to the

initial composition of 1% solvent B. A LTQ XL (Thermo, San Jose, CA) was used to

perform tandem mass spectrometry using data dependent acquisition method selecting

the top 10 most abundant ions using Xcaliber 2.0.7 (Thermo). The analysis was set up

for a full scan recorded between m/z 350 – 2000, and a MS/MS scan to generate

product ion spectra to determine amino acid sequence in consecutive instrument scans

74

of the ten most abundant peaks in the spectrum. The method was set up to perform full-

MS and subsequent MS/MS scan on the top 10 most intense ions from the full MS scan

spectrum with dynamic exclusion. The dynamic exclusion puts a mass in the exclusion

list once the MS/MS is acquired and subsequently performs MS/MS on the second most

intense ion. Dynamic exclusion was enabled with a repeat count of 2 within 30 seconds,

a mass list size of 50, an exclusion duration 30 seconds, the low mass width was 0.5

and the high mass width was 1.5.

The MS/MS data was analyzed using Proteome Discoverer 1.3 (Thermo) to

search against Uniprot human database version 2014_09 (85,838 sequences and

33,867,335 residues) with no enzyme. Unlike trypsin that has a known cleavage site; at

the carboxyl side of the amino acids lysine or arginine, calpain does not have a known

cleavage site. Because of this reason the data was searched with no enzyme as option.

The search was achieved using the average mass for matching the precursor with a

fragment ion mass tolerance of 0.8 Da and a precursor ion tolerance of 0.8 Da.

carbamidomethylation of cysteine was selected as a static modification, while the

oxidation of methionine was selected as a dynamic modification. Two missed cleavages

for the enzyme were permitted.

Results and Discussion

Mass spectrometry

The filtrate from the 10-kDa MWCO filter was analyzed by mass spectrometry.

This filtrate consisted of smaller proteolytic peptides and proteins following TBI. The

CSF samples were analyzed similar to the brain lysate samples. MS/MS was performed

on the top 10 most abundant ions from MS spectra. The resulting MS/MS data collected

was searched using Proteome Discoverer database search. In this search the

75

fragmentation spectra were compared to the theoretical fragmentation in the database,

which then predicts the sequence of the peptides. The database then generates a list of

possible proteins from the identified peptides. From this list, the proteins with a score of

1.5 and higher were selected. This list of possible hits from the database search was

extensive. The list of proteins in the TBI samples was then compared to the proteins

identified in the control samples. The proteins unique to control sample only, unique to

TBI samples and common in both control and TBI were prepared using excel. Using this

data, a Venn diagram (Figure 3-1) was constructed which shows the average hits for

the control samples and average hits for all the TBI CSF samples with a number of hits

common in both control and TBI samples. By using the list of proteins unique in TBI

samples, all the brain specific proteins were selected. Also, the proteins that were

subjected to calpain cleavage by performing in vitro digestion in mouse brain lysate and

CCI mouse brain lysate in the previous chapter were also selected. There were few

proteins that were subjected to proteolysis following brain injury, which were observed

in vitro digestion, mouse CCI samples and also in the human TBI-CSF samples. Table

3-1 shows this list of proteins identified in the human TBI CSF samples. This list of

proteins showed a match of atleast two peptides from the database search result. The

low molecular weight proteins, neurogranin and MBP were observed in the TBI-CSF

samples by mass spectrometry. The peptides identified from the database search

results for these two proteins were mapped and MS/MS spectra for these peptides were

obtained. Human MBP isoform sequence with identified peptides is shown in Figure 3-2.

The MS/MS spectrum for one of the MBP peptide, -HGSKYLATASTMD with the

identified fragment ions is shown in Figure 3-3. The identified fragment ions for the other

76

MBP peptides –TQDENPVVHF from the database are shown in Figure 3-4. Similarly,

human neurogranin sequence with identified peptides mapped is shown in Figure 3-5.

MS/MS spectrum with identified fragment ions for this peptide is shown in Figure 3-6.

Western blot

Western blot was performed on the samples that were retained in the 10-kDa

MWCO filter after filtration. Western blot was used to confirm proteolysis of proteins in

the TBI samples. The antibodies used to probe the membrane were alpha spectrin,

GFAP and MBP. These antibodies were selected based on the proteins identified by the

mass spectrometry data and their availability. In all the TBI samples, the spectrin was

significantly degraded to 150 kDa. The blots probed with spectrin antibody are shown in

Figure 3-7(A). The color density of the intact and breakdown product was quantified and

plotted against arbitrary density units (Figure 3-7 B). Since spectrin was also observed

by mass spectrometry, detection of spectrin breakdown products by western blot

confirms that spectrin is subjected to proteolytic breakdown following brain injury.

Spectrin breakdown products were also observed in the in vitro digestion of mouse

brain lysate and CCI samples as discussed in the previous chapter.

Similarly GFAP breakdown product (38 kDa) was also observed in all the TBI

samples. The blots probed with GFAP antibody are shown in Figure 3-8(A). The GFAP

breakdown products showed an increase in the TBI samples (Figure 3-8B). CSF sample

from patient 5 and patient 10 showed large amounts of albumin that made the gel and

blot skewed. This made it difficult to quantitate the breakdown products. Therefore for

patient 10 the GFAP density was not collected. The presence of GFAP breakdown

products thus shows that GFAP is also subjected to breakdown following brain injury.

77

The MBP antibody available probed the breakdown products of MBP. The MBP

breakdown product was observed at 10-kDa in all the TBI samples. The MBP

breakdown product was not observed in patients 7-12. In patient number 10 due to

skewed lane it was difficult to confirm the presence of MBP breakdown product. Figure

3-9 (A) shows the image of the blots probed with MBP antibody and Figure 3-9 (B)

shows the quantified data of MBP breakdown products. MBP was also detected by

mass spectrometry data. Western blot validates the proteolysis of MBP following the

brain injury.

Neurogranin antibody was also used to probe the blots with TBI samples.

Neurogranin was observed in only three TBI patient’s CSF samples. Out of these three

patients, neurogranin was observed in one patient by mass spectrometry as well. Figure

3-10 (A) and (B) displays the image of the blots probed with neurogranin antibody and

the quantified neurogranin band in the TBI CSF samples.

Conclusions

This work demonstrated that possible TBI biomarkers could be identified from the

clinical samples like cerebrospinal fluid. Since CSF is in close contact with the brain and

spinal cord, it makes it an ideal biological fluid to study neurological changes or

diseases. By filtering the CSF through the 10-kDa MWCO filter, the proteolytic peptides

and protein smaller than 10-kDa were separated. By performing MS/MS on this filtrate

and using Proteome Discoverer database search to predict the proteins there were few

proteins identified that could possible serve as biomarkers for brain injury. Some of

these proteins were also observed by in vitro calpain digestion and CCI samples of the

mouse brain that was discussed in the previous chapter. The list of proteins shown in

78

Table 3-1 were selected based on brain specific proteins, had good sequence

coverage, and from the in vitro digestion of mouse brain lysates.

To validate the MS/MS data, western blot was performed on the samples that

were retained on the membrane filter. Using western blot some proteins; spectrin,

GFAP and MBP were confirmed that these proteins are subjected to proteolysis

following brain injury. The breakdown products of these proteins showed increase in all

the TBI samples. More proteins can be validated by western blot provided there is

availability of specific antibodies for each protein.

Although, collection of CSF is an invasive technique, it is an ideal biofluid to

study the brain injury biomarkers. Identification of biomarkers from human biofluid like

CSF can be useful in diagnosing the severity of the injury and further provide better

prognosis of the disease and further aid in better treatment by the physician.

79

Figure 3-1. Venn diagram showing the number of proteins identified using Proteome Discoverer based on the LC-MS/MS data from human clinical TBI CSF samples. These data are based on the filtrate from filtration through the 10,000 Da MWCO filter.

Control

2085

TBI

1675472

80

Table 3-1. List of protein biomarkers identified in human CSF samples by LC-MS/MS.

Accession number

Full protein Name Gene Name Molecular weight (kDa)

Q02410 Amyloid beta A4 precursor protein-binding family A member 1 APBA1 92.8 Q5T5Y3 Calmodulin regulated spectrin-associated protein 1 CAMSAP1 177.9 Q9P1Y5 Calmodulin-regulated spectrin-associated protein 3 CAMSAP3 134.7 Q6UVK1 Chondroitin sulfate proteoglycan 4 CSPG4 91.7 P14136 Glial fibrillary acidic protein GFAP 49.8 P42858 Huntingtin HTT 347.1 P11137 Microtubule-associated protein 2 MAP2 199.4

Q96JE9 Microtubule-associated protein 6 MAP6 86.5 P07196 Neurofilament light polypeptide NEFL 61.5 Q8NF91 Nesprin-1 SYNE1 1010.5 Q92686 Neurogranin NRGN 7.6 P02686 Myelin basic protein MBP 33.1 A6NG51 Spectrin alpha chain, nonerythrocytic 1 SPTAN1 284.8 O43815 Striatin STRN 86.1 P17600 Synapsin I SYN1 74.1

O14994 Synapsin III SYN3 63.3 P07437 Tubulin beta TUBB 49.6

81

1 MGNHAGKREL NAEKASTNSE TNRGESEKKR NLGELSRTTS EDNEVFGEAD

51 ANQNNGTSSQ DTAVTDSKRT ADPKNAWQDA HPADPGSRPH LIRLFSRDAP

101 GREDNTFKDR PSESDELQTI QEDSAATSES LDVMASQKRP SQRHGSKYLA

151 TASTMDHARH GFLPRHRDTG ILDSIGRFFG GDRGAPKRGS GKDSHHPART

201 AHYGSLPQKS HGRTQDENPVVHFFKNIVTP RTPPPSQGKG RGLSLSRFSW

251 GAEGQRPGFG YGGRASDYKS AHKGFKGVDA QGTLSKIFKL GGRDSRSGSP

301 MARR

Figure 3-2. Human MBP isoform sequence with mapped peptides identified from

database search results.

82

A

B

b⁺ b²⁺ Seq. y⁺ y²⁺

1 138.14879 69.57809 H 13

2 195.20049 98.10394 G 1245.39679 623.20209 12

3 282.27849 141.64294 S 1188.34509 594.67624 11

4 410.45299 205.73019 K 1101.26709 551.13724 10

5 573.62960 287.31850 Y 973.09259 487.04999 9

6 686.78940 343.89840 L 809.91598 405.46169 8

7 757.86810 379.43775 A 696.75618 348.88179 7

8 858.97320 429.99030 T 625.67748 313.34244 6

9 930.05190 465.52965 A 524.57238 262.78989 5

10 1017.12990 509.06865 S 453.49368 227.25054 4

11 1118.23500 559.62120 T 366.41568 183.71154 3

12 1249.43440 625.22090 M 265.31058 133.15899 2

13 D 134.11118 67.55929 1

Figure 3-3. MS/MS spectra for the MBP peptide HGSKYLATASTMD. (A) MS/MS

spectra displaying the fragment ions for this peptide. (B). Identified b- and y-type ions for the MBP peptide HGSKYLATASTMD shown in red and blue from the database search results.

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757.93504.39541.91 858.92

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821.86720.01 1118.66 1245.16949.57 1188.481028.59879.93403.50366.52265.24233.811362.66

y12 y11

y6

y3 y2

b8 b9 b10

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83

b⁺ b²⁺ Seq. y⁺ y²⁺

1 102.11249 51.55994 T 10

2 230.24340 115.62540 Q 1085.16259 543.08499 9

3 345.33190 173.16965 D 957.03168 479.01954 8

4 474.44750 237.72745 E 841.94318 421.47529 7

5 588.55140 294.77940 N 712.82758 356.91749 6

6 685.66820 343.33780 P 598.72368 299.86554 5

7 784.80100 392.90420 V 501.60688 251.30714 4

8 883.93380 442.47060 V 402.47408 201.74074 3

9 1021.07520 511.04130 H 303.34128 152.17434 2

10 F 166.19988 83.60364 1

Figure 3-4. Identified b- and y-type ions for the MBP peptide TQDENPVVHF shown in

red and blue.

84

1 MDCCTENACS KPDDDILDIP LDDPGANAAA AKIQASFRGH MARKKIKSGE

51 RGRKGPGPGGPGGAGVARGGAGGGPSGD

Figure 3-5. Human neurogranin sequence with the mapped peptide identified from

database search results.

85

A

B

b⁺ b²⁺ Seq. y⁺ y²⁺

1 58.05909 29.53324 G 19

2 155.17589 78.09164 P 1349.44698 675.22719 18

3 212.22759 106.61749 G 1252.33018 626.66879 17

4 269.27929 135.14334 G 1195.27848 598.14294 16

5 366.39609 183.70174 P 1138.22678 569.61709 15

6 423.44779 212.22759 G 1041.10998 521.05869 14

7 480.49949 240.75344 G 984.05828 492.53284 13

8 551.57819 276.29279 A 927.00658 464.00699 12

9 608.62989 304.81864 G 855.92788 428.46764 11

10 707.76269 354.38504 V 798.87618 399.94179 10

11 778.84139 389.92439 A 699.74338 350.37539 9

12 935.02939 468.01839 R 628.66468 314.83604 8

13 992.08109 496.54424 G 472.47668 236.74204 7

14 1049.13279 525.07009 G 415.42498 208.21619 6

15 1120.21149 560.60944 A 358.37328 179.69034 5

16 1177.26319 589.13529 G 287.29458 144.15099 4

17 1234.31489 617.66114 G 230.24288 115.62514 3

18 1291.36659 646.18699 G 173.19118 87.09929 2

19 P 116.13948 58.57344 1

Figure 3-6. MS/MS spectra for the neurogranin peptide GPGGPGGAGVARGGAGGGP. (A) MS/MS spectra displaying the fragment ions for this peptide. (B). Identified b- and y-type ions for the neurogranin peptide shown in red and blue identified from the database search results.

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934.98523.66 560.56423.56 799.15 984.06462.46 820.81497.89 878.05760.09 1041.07727.81 1112.26

b6 b9 b12

b15

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GPGGPGGAGVARGGAGGGP

86

Figure 3-7. Western blot of human TBI-CSF samples, that were retained on the

10,000Da molecular weight cut off (MWCO) filter after filtration and probed with alpha-spectrin antibody. (A) Western blot displaying the breakdown product of spectrin observed in the TBI-CSF samples. (B) Quantitation of breakdown product of alpha-spectrin. Error bars represent the standard error of the mean (N=12). * denotes SBDP 150 for TBI more than control (P=0.0324)

A

SBDP (150 kDa)

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40

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100

120

Control TBI

Arb

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y D

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87

Figure 3-8. Western blot of human TBI-CSF samples, that were retained on the

10,000Da molecular weight cut off (MWCO) filter after filtration and probed with GFAP antibody. (A) Western blot displaying the breakdown product of GFAP observed in the TBI-CSF samples. (B) Quantitation of breakdown product of GFAP. Error bars represent the standard error of the mean (N=12). * Denotes GFAP-BDP for TBI more than control (P=0.0074).

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GFAP (52 kDa)

B

0

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Arb

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Figure 3-9. Western blot of human TBI-CSF samples, that were retained on the

10,000Da molecular weight cut off (MWCO) filter after filtration and probed with MBP antibody. (A) Western blot displaying the breakdown product of MBP observed in the TBI-CSF samples. (B) Quantitation of breakdown product of MBP. Error bars represent the standard error of the mean (N=12). * Denotes MBP-BDP for TBI more than control (P=0.0337).

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Figure 3-10. Western blot of human TBI-CSF samples, that were retained on the

10,000Da molecular weight cut off (MWCO) filter after filtration and probed with neurogranin antibody. (A) Western blot displaying the intact neurogranin observed in the TBI-CSF samples. (B) Quantitation of intact neurogranin. Error bars represent the standard error of the mean (N=12).

Neurogranin

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90

CHAPTER 4 SYSTEMS BIOLOGY APPROACH TRAUMATIC BRAIN INJURY BIOMARKERS

Introduction

Systems biology has been considered the latest domain in the biological

sciences that aims for a systems-level understanding of complex biological processes.

[50] Systems biology application explains the function of molecules and biological

structures such as cells, tissue and organelles, in a cellular component in normal or

perturbed conditions. [51] The ultimate goal of systems biology is; to explore the system

to help biologists, aid pharmaceutical companies and doctors to better understand the

mechanism underlying the disease and further find suitable targets for the treatment.

With this approach, one can predict the functions and behavior of various components

of the system. [52, 53] Since traumatic brain injury is the outcome of complex biological

systems responses rather than one individual gene, protein or pathway, system biology

approach in the analysis of these networks can give important clues of the underlying

processes. [53] This can help in potential improvements in therapeutic discovery. System

biology is a combination of experimental, proteomics, and genomics datasets, literature

and text mining, bioinformatics and pathway/interaction mapping methods. [53]

Pathway analysis

With the rapid growth of high-throughput technology in genomic and proteomic

studies, a tremendous amount of data has been generated. The molecular pathway

provides wiring diagrams describing how the gene products, and other biomolecules

interact, relate, and regulate with each other to perform particular biological functions.

[54, 55] Pathways are manually curated from the literature into large online datasets,

which can be used to link disease or injury specific genes to biological processes and

91

identify pathways associated with the studied disease or injury condition. [56] A

comprehensive database of TBI-related information, including high-throughput “omic”

datasets (genomics, proteomics, metabolomics, etc.) and “targeted” pathway,

pharmacology, and molecular imaging studies from the relevant scientific literature can

be developed. There are various model representations to serve different purposes.

Pathway Studio, Ingenuity Pathway, and Gene GO are used to generate graphical

diagrams of biological processes to provide a visual presentation of network models by

incorporating genome, proteome, and metabolome data. [53] Systems biology tools are

unbiased approaches to identify non-redundant neuromodulation/neurotoxicity

pathways and to further pinpoint candidate diagnostic biomarkers and/or molecular

targets for therapy. These tools help to organize available data and knowledge

according to the high-level, holistic view required by system biology. [56]

Experimental

A list of protein biomarkers identified by performing in vitro digestion of naïve

brain lysate by calpain and from the animal TBI model samples in project 1 was used to

study interactions between these proteins and how are they involved in biological cell

processes and they regulate in diseases. Pathway Studio software 9.0 (Ariadne

Genomics Inc. MD, USA) was used to identify significant traumatic brain injury

biomarkers pathways. List of the identified protein biomarkers used is shown in Table 4-

1.

Results and Discussion

Pathway studio 9.0 was used to generate map of protein-protein interaction as

shown in Figure 4-1. The network was generated using the direct links algorithm to map

the regulation of each protein with each other. The nodes in the network denote the

92

proteins and the arrows connecting the proteins shows the regulation between each

proteins. The regulations are based on the literature mining. For example, the proteins

MAP1B and MAP1A are connected to each since they are expressed together in the

neurite formation and stabilization. It is shown that the levels of MAP1A and MAP1B

change dramatically during development, with MAP1B expression highest in forming

neurons, and MAP1A expression highest in mature neurons. [57] In the other example,

GFAP and protein neurofilament light polypeptide (NEFL) shows positive regulation with

each other. One of the references discusses that NEFL, and GFAP are specific

markers for damage of the central nervous system and increased concentrations

of NFL and GFAP have previously been found both after acute and chronic brain

injuries caused by different types of trauma. [58]

Using the same protein biomarkers, but different algorithm; common targets with

cell process and diseases, regulation between these proteins were mapped in the

Pathway studio software. Figure 4-2 shows the map of how these proteins regulate cell

processes and diseases. These maps are generated based on the literature published

on these proteins. For example, the map shows how myelin basic protein (MBP) is

involved in cell processes like myelination and remyelinization of myelin sheath and is

also regulated in diseases like TBI, demyelination, nerve injury, Alzheimer’s, etc. The

connector between proteins, cell processes and diseases show the references how they

have been linked to each other. For example, it has been reported that myelin basic

protein is involved in the myelination of nerves in the central nervous system. [59] Also,

myelin basic protein is essential for the formation of the myelin sheath. [60] Similarly,

there the other proteins are mapped showing how they are involved in the cell

93

processes and diseases. Thus with help of these mappings one can understand the

pathological pathways of proteins.

Conclusions

A system biology approach was used to study the regulation of proteins in

biological cell processes and diseases. Pathways of brain injury potential biomarkers,

identified in chapter 2 and 3, were generated using Pathway Studio 9.0 software. It

showed the protein-protein interactions between the protein biomarkers and also how

these biomarkers are involved in biological cell processes and diseases by showing the

regulation between them. This approach can further help in better diagnosis and

development of targeted treatment by the physician and pharmaceutical companies.

94

Table 4-1. List of brain injury protein biomarkers entities

Name Description

BASP1 Brain abundant, membrane attached signal protein 1

CAMSAP2 Calmodulin regulated spectrin-associated protein family, member 2

CAMSAP3 Calmodulin regulated spectrin-associated protein family, member 3

CSPG4 Chondroitin sulfate proteoglycan 4

CSPG5 Chondroitin sulfate proteoglycan 5

CKB Creatine kinase, brain

GFAP Glial fibrillary acidic protein

GAD1 Glutamate decarboxylase 1

HTT Huntingtin

MAST1 Microtubule associated serine/threonine kinase 1

MAP1A Microtubule-associated protein 1A

MAP1B Microtubule-associated protein 1B

MAP2 Microtubule-associated protein 2

MAP6 Microtubule-associated protein 6

MAPT Microtubule-associated protein tau

MBP Myelin basic protein

NRXN1 Neurexin 1

NRXN2 Neurexin 2

NRXN3 Neurexin 3

NCAN Neurocan

NFASC Neurofascin

NEFH Neurofilament, heavy polypeptide

NEFL Neurofilament, light polypeptide

NEFM Neurofilament, medium polypeptide

NRGN Neurogranin (protein kinase C substrate, RC3)

SPTA1 Spectrin, alpha, erythrocytic 1

SPTBN2 Spectrin, beta, non-erythrocytic 2

SPTBN4 Spectrin, beta, non-erythrocytic 4

SYN1 Synapsin I

SYN2 Synapsin II

SYN3 Synapsin III

SNCB Synuclein, beta

TUBB4B Tubulin, beta 2C

TUBB4A Tubulin, beta 4

USP1 Ubiquitin specific peptidase 1

95

Figure 4-1. Mapping of protein biomarkers showing direct interaction pathway.

96

Figure 4-2. Mapping of protein biomarkers showing regulations of cell process and diseases.

97

CHAPTER 5 SUMMARY AND FUTURE DIRECTIONS

Summary

To gain a comprehensive understanding of the breakdown of proteins following

brain injury, there is a need to study the pathophysiology of the head injury for adequate

diagnosis and treatment. One of the pathophysiological mechanisms is known to involve

degradation of proteins by calpain due to increased concentration of Ca2+ following

injury to the brain. In this work, brain injury biomarkers were studied by performing in

vitro digestion of the brain lysate using calpain in presence of calcium. The calpain

cleaved proteins were identified by LC-MS/MS and immunoblotting methods.

Mass spectrometry based proteomics is a promising technique due to the

sensitivity of protein identification and characterization. In this study, mass spectrometry

based proteomics was used to identify the brain injury protein biomarkers. After

performing in vitro calpain digestion, the samples were analyzed by LC-MS/MS. The

MS/MS data was then processed using Proteome Discoverer search program. From the

database search hits, few potential protein breakdown biomarkers were identified.

These biomarkers were then searched in the CCI mouse brain model. There were some

proteins found common in the CCI mouse brain lysates. A similar study involving clinical

samples of human cerebrospinal fluid from patients suffering from TBI were used to find

the protein breakdown products. A few possible protein biomarkers were found by using

a similar methodology for the animal model. Table 5-1 shows the proteins identified by

in vitro digestion, in CCI samples and in human TBI CSF samples. This Table shows

that some proteins were observed in all the three models.

98

Lastly, a system biology approach was used to determine the biochemical

pathway involved in the pathogenesis of brain injury based on the protein biomarkers

identified by in vitro digestion of brain lysate, animal TBI model, and human clinical CSF

samples. This study of biology at the system level will help revolutionize our

understanding of complex biological regulatory system and provide new opportunities

for practical application of such knowledge. The most feasible application of system

biology research is to create a detailed model of cell regulation that will provide insights

into mechanism based drug discovery. Some of the protein degradome and its

localization are displayed in Figure 5-1.

Future Work

Future studies will investigate a different pathophysiological pathway involved in

TBI. In this study we focused on calpain cleavage proteolysis of brain proteins. But

there are other proteolysis cleavages involved in TBI. One of the other proteolytic

pathways involves breakdown of proteins by caspases. It can also be studied using the

same method used in this research.

There is also a scope to study the identified protein biomarkers by western blot.

Since western blot involves use of protein specific antibody, it will aid in validation of

these identified biomarkers. Western blot is a powerful procedure for immunoblotting of

proteins, even if they are present in low abundance in a given sample.

Future studies will also aim to study TBI biomarkers in other biological fluids like

blood. Since withdrawing of blood is less invasive compared to the procedure involved

for collection of CSF. Analyzing blood sample would also be easier if there would be

development of hand-held devices for diagnoses of TBI like that of a blood glucose

99

monitor. Furthermore, there is more scope to expand the systems biology approach,

where it can be used to study other diseases involved in central nervous system injury.

100

Table 5-1. Summary of biomarkers identified by invitro digestion, mouse CCI samples, and human TBI CSF samples.

In Vitro Digestion

Proteins Calcium only

Calpain CCI TBI

Amyloid beta A4 precursor protein-binding family B member 1-interacting protein

+ +

Brain soluble acidic protein 1 + + +

Calmodulin regulated spectrin-associated protein 1 + Calmodulin regulated spectrin-associated protein 2 + +

Calmodulin regulated spectrin-associated protein 3 + + +

Chondroitin sulfate proteoglycan 4 + + + Creatine kinase B-type + Glial fibrillary acidic protein + Glutamate decarboxylase 1 + +

Huntingtin + + + +

Microtubule-associated protein 1A + + + Microtubule-associated protein 1B + + + Microtubule-associated protein 2 + + + + Microtubule-associated protein 6 + + + + Microtubule-associated serine/threonine-protein kinase 1

+

Myelin basic protein + + + + Nesprin-1 + Neurexin-1 + +

Neurexin-3 + +

Neurocan + + Neurochondrin + Neurofascin + + Neurofilament heavy polypeptide + +

Neurofilament light polypeptide + + +

Neurofilament medium polypeptide + +

Neurogranin + + Secretogranin-2 + Spectrin alpha chain, erythrocytic 1 + + + + Striatin + + Synapsin I + + Synapsin II + + + Synapsin III + + Synaptotagmin-2 +

Synuclein, beta +

Tubulin beta-2A chain + +

Tubulin beta-4A chain +

101

Figure 5-1. Protein degradome and its localization.

Neuron Cell Body: BASP1 Neuronal Axons: αII-

Spectrin, βII-Spectrin, CAMSAP1, 3 NEFL, NEFH

Astroglial cells: GFAP

Neuronal Synaptic Terminal: Neurogranin, Synapsin, CaMPK-II

Oligodendrocytes (myelin): MBP

Neuronal Dendrites: MAP2, MAP1A/B, MAP4

102

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BIOGRAPHICAL SKETCH

Manasi N. Kamat, daughter of Anuradha and Dilip Mangaonkar and a younger

sibling of Mahesh Mangaonkar, was born and raised in Mumbai, India. Manasi received

her Bachelor of Science in life sciences at the University of Mumbai, India. Manasi also

did her Master of Science in bioanalytical sciences at the University of Mumbai. As a

summer intern, Manasi worked at Rubicon, a pharma based company in Mumbai, India.

She worked on a forced degradation study of formulations. After receiving her MS she

worked at SITEC, Cipla in Mumbai, India, in a bioanalytical department. She worked on

developing methods for extraction of analytes from blood plasma for clinical trial studies.

She then married Naren Kamat and moved to Gainesville, Florida. She decided

to continue with her studies at the University of Florida (UF) and joined the chemistry

department as a graduate student in 2010. In the summer of 2012, Manasi graduated

with a Master of Science in analytical chemistry. Manasi did her MS research in the field

of mass spectrometric imaging to study the effect of traumatic brain injury on the

proteins in the brain, under the direction of Dr. David Powell, Dr. Richard Yost and Dr.

Kevin Wang. She has a beautiful daughter, Zoey, born in 2013. She enjoys spending all

her free time playing with her. After her MS, Manasi decided to work towards her

doctoral studies by continuing her work with Dr. Richard Yost and Dr. Kevin Wang at the

University of Florida. Her research was on identifying traumatic brain injury biomarkers

from animal model and clinical samples. Manasi completed her doctorate in December

2015.